Participación de los sistemas Toxina-Antitoxina de Tipo II y proteasas Clp de C/ostridioides difficile R20291 en la generación de células persistentes y en fenotipos virulentos
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Fecha
2020
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Profesor/a Guía
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Idioma
es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
En las últimas décadas, se ha evidenciado un aumento en la frecuencia de
patógenos bacterianos que tiene multirresistencia a fármacos y tolerancia a antibióticos.
Los mecanismos clásicos de resistencia a antibióticos en patógenos bacterianos son
generalmente causados por mutaciones o adquisición de genes de resistencia. Por el
contrario, los mecanismos de tolerancia implican la aparición de células persistentes, las
cuales son variantes fenotípicas de bacterias silvestres que contribuyen a la persistencia
bacteriana en el hospedero. Estas células se generan principalmente frente a un
ambiente estresante, pero también pueden generarse de forma estocástica. El
mecanismo por el cual se forman ha sido difícil de dilucidar debido a que solo una
pequeña fracción de la población se vuelve persistente, pero un factor común son las
toxinas de los sistemas toxina-antitoxina (TA) y las proteasas Lon y Clp.
Los sistemas TA están compuestos por dos genes, los que codifican para una
toxina y su antitoxina afín. En condiciones normales de crecimiento, la antitoxir.a se
forma un complejo con la toxina, lo que evita que esta última ejerza su efecto. Mientras
que, frente al estrés ambiental causado por los antibióticos, se genera una baja en los
niveles de la antitoxina, debido a su degradación por proteasas propias de la bacteria
(Lon y Clp), produciéndose la liberación y activación de la toxina, lo que llevará a la
inhibición del crecimiento y, por lo tanto, a la generación de células persistentes. Los
sistemas TA han sido relacionados con la persistencia bacteriana en muchos patógenos
bacterianos. Sin embargo, tanto la persistencia como la contribución de los sistemas TA
de Tipo II y las proteasas Lon y Clp, no han sido estudiados en el patógeno nosocomial
Clostridioides difficile. Este patógeno es el agente etiológico de las infecciones causadas
por C. difficile, las cuales son responsables del 30% de las diarreas infecciosas asociadas
al uso de antibióticos. Además, tiene gran capacidad de persistir en el hospedero, lo que
contribuye a la alta tasa de infecciones recurrentes.
En este trabajo determinamos que C. difficile forma células persistentes frente
al tratamiento con antibióticos in vitro. Además, C. difficile R20291 posee 9 sistemas TA
de Tipo II hipotéticos, de los cuales RelBE y MazEF son funcionales, poseen actividad
endorribonucleasa y aumentan su expresión frente a vancomicina, la que es dependiente
de la proteasa ClpP2 y la chaperona ClpC. Finalmente, ClpP2 y ClpC participan en
formación de células persistentes, en la generación de biopelículas, motilidad,
producción de toxinas, esporulación y adherencia a células epiteliales. Estos resultados
sugieren que ClpP2 y ClpC contribuyen a la virulencia de C. diffici/e R20291.
In recent decades, there has been an lncrease in the frequency of bacteria! pathogens that has multidrug resistance and antibiotic tolerance. The classic mechanisms of antibiotic resistance in bacteria! pathogens are generally ca u sed by mutations or acquisition of resistan ce genes. In contrast, toler3nce mechanisms involve the appearance of persister cells, which are phenotypic variants of wild type bacteria that contribute to persistence in the host. These cells are generated primarily in a stressful environment, but they can also be generated stochastically. The mechanism by which they are formed has been difficult to elucidate because only a small fraction of the population becomes persister, but a common factor is toxins of the toxin antitoxin (TA) systems, and Lon and Clp proteases. TA systems are composed of two genes, which code for a toxin and its related antitoxin. Under normal growth conditions, the antitoxin forms a complex with the toxin, which prevents the latter from exerting its effect. In contrast, against environmental stress caused by antibiotics, a decrease in antitoxin levels is generated, due to its degradation by bacteria! proteases (Lon and Clp), resulting in the release and activation of the toxin, which it will lead to growth inhibition and, therefore, to the generation of persister cells. TA systems have been related to bacteria! persistence in many bacteria! pathogens. However, both the persistence and the contribution of Type II TA systems and Lon and Clp proteases have not been studied in the nosocomial pathogen Clostridioides difficile. This pathogen is the etiologic agent of infections caused by C. diffici/e, which are responsible for 30% of infectious diarrhea associated with the use of antibiotics. In addition, it has a great capacity to persist in the host, which contributes to the high rate of recurrent infections. In this work we determine that C. difficife generates persister cells against antibiotic treatment in vitro. In addition, C. difficile R20291 has 9 hypothetical Type 11 TA systems, of which RelBE and MazEF are functional, possess endoribonuclease activity and increase their expression against vancomycin, which is dependent on the ClpP2 protease and the ClpC chaperone. Finally, ClpP2 and ClpC participate in the formation of persister cells, in the generation of biofilms, motility, toxin production, sporulation, and adhesion to epithelial cells. These results suggest that ClpP2 and ClpC contribute to the virulence of C. difficile R20291.
In recent decades, there has been an lncrease in the frequency of bacteria! pathogens that has multidrug resistance and antibiotic tolerance. The classic mechanisms of antibiotic resistance in bacteria! pathogens are generally ca u sed by mutations or acquisition of resistan ce genes. In contrast, toler3nce mechanisms involve the appearance of persister cells, which are phenotypic variants of wild type bacteria that contribute to persistence in the host. These cells are generated primarily in a stressful environment, but they can also be generated stochastically. The mechanism by which they are formed has been difficult to elucidate because only a small fraction of the population becomes persister, but a common factor is toxins of the toxin antitoxin (TA) systems, and Lon and Clp proteases. TA systems are composed of two genes, which code for a toxin and its related antitoxin. Under normal growth conditions, the antitoxin forms a complex with the toxin, which prevents the latter from exerting its effect. In contrast, against environmental stress caused by antibiotics, a decrease in antitoxin levels is generated, due to its degradation by bacteria! proteases (Lon and Clp), resulting in the release and activation of the toxin, which it will lead to growth inhibition and, therefore, to the generation of persister cells. TA systems have been related to bacteria! persistence in many bacteria! pathogens. However, both the persistence and the contribution of Type II TA systems and Lon and Clp proteases have not been studied in the nosocomial pathogen Clostridioides difficile. This pathogen is the etiologic agent of infections caused by C. diffici/e, which are responsible for 30% of infectious diarrhea associated with the use of antibiotics. In addition, it has a great capacity to persist in the host, which contributes to the high rate of recurrent infections. In this work we determine that C. difficife generates persister cells against antibiotic treatment in vitro. In addition, C. difficile R20291 has 9 hypothetical Type 11 TA systems, of which RelBE and MazEF are functional, possess endoribonuclease activity and increase their expression against vancomycin, which is dependent on the ClpP2 protease and the ClpC chaperone. Finally, ClpP2 and ClpC participate in the formation of persister cells, in the generation of biofilms, motility, toxin production, sporulation, and adhesion to epithelial cells. These results suggest that ClpP2 and ClpC contribute to the virulence of C. difficile R20291.
Notas
Tesis (Doctor en Biotecnología)
Palabras clave
Clostridium Difficile, Resistencia a los Antibióticos, Proteínas