Estudio del efecto del restablecimiento en cis del gen iraP en Salmonella Typhi STH2370 con respecto a la acumulaciĆ³n de RpoS y posibles fenotipos asociados
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2017
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es
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Universidad AndrƩs Bello
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Salmonella Typhi (S. Typhi), agente causal de la fiebre tifoidea, infecta exclusivamente al ser humano. A pesar de que se encuentra estrechamente relacionada con Salmonella Typhimurium (S. Typhimurium), difieren en la especificidad de hospedero. Estas diferencias responden a un proceso evolutivo multifactorial que depende de la ganancia de funciones, la perdida de funciones y la regulaciĆ³n diferencial de los genes compartidos entre estos dos serovares. Un ejemplo de regulaciĆ³n diferencial es la ejercida sobre el factor sigma alternativo, RpoS, regulador maestro de la respuesta general a estrĆ©s, que contribuye en el ciclo infectivo de estos patĆ³genos. Este factor estĆ” regulado a distintos niveles, siendo los mecanismos de proteĆ³lisis los mĆ”s importantes. PhoPQ es un sistema de dos componentes que regula positivamente la acumulaciĆ³n de RpoS en S. Typhimurium. En estrĆ©s por bajas concentraciones de Mg2+, PhoP activa la transcripciĆ³n del gen que codifica a IraP, proteĆna que impide que RpoS sea degradado por ClpXP. En nuestro laboratorio se ha observado que la acumulaciĆ³n de RpoS es diferente entre S. Typhimurium y S. Typhi, determinĆ”ndose que la acumulaciĆ³n de RpoS en S. Typhi aumenta en mutantes ĪphoPQ. Como estudios previos se realizĆ³ un anĆ”lisis bioinformĆ”tico de S. Typhi y S. Typhimurium para intentar explicar estas diferencias y se encontrĆ³ que nuestra cepa de trabajo S. Typhi STH2370 perdiĆ³ un segmento genĆ³mico en la que estaba incluido el gen iraP. Por lo que nos preguntamos si la falta de iraP en S. Typhi es responsable de las diferencias en la acumulaciĆ³n de RpoS al compararse con S. Typhimurium. En la presente Tesis, se incorporĆ³ el gen iraP de S. Typhimurium en S. Typhi STH2370 en una Ćŗnica copia situada en su contexto genĆ©tico original mediante una tĆ©cnica de complementaciĆ³n heterĆ³loga en cis y se evaluĆ³ su rol en la acumulaciĆ³n de RpoS en la cepa silvestre y en mutantes ĪphoPQ, utilizando fusiones lacZ,
Western blot, ensayos de estabilidad de proteĆnas y ensayos de supervivencia a perĆ³xido de hidrĆ³geno. AdemĆ”s, se realizaron ensayos de invasiĆ³n y proliferaciĆ³n en cĆ©lulas epiteliales HT-29 para determinar si la ausencia de iraP corresponde a un caso de pĆ©rdida de funciĆ³n que tiene implicancia en la virulencia de S. Typhi. Encontramos que IraP incrementa la acumulaciĆ³n y la estabilidad de RpoS en condiciones de bajo Mg2+. Por otra parte, la presencia de IraP cambia la regulaciĆ³n de RpoS mediada por PhoPQ. Finalmente se observĆ³ que la cepa complementada con iraP presentĆ³ una disminuciĆ³n en la capacidad de invasiĆ³n de cĆ©lulas epiteliales HT-29 en comparaciĆ³n con la cepa silvestre. Los resultados obtenidos sugieren que la pĆ©rdida de iraP en S. Typhi podrĆa implicar ciertas ventajas evolutivas.
Salmonella Typhi (S. Typhi), the causative agent of typhoid fever, exclusively infects humans. Although S. Typhi is closely related to Salmonella Typhimurium (S. Typhimurium), they differ in host specificity. These differences are due to a multifactorial evolutive process that depends on gain functions, loss functions, and a differential regulation of the genes shared by both serovars. An example of the latter is the regulation of the alternative Sigma factor, RpoS, a master regulator of stress response, that contributes to the infective cycle of these pathogens. This Sigma factor is regulated at different levels, being proteolysis one of the most important regulation mechanisms. PhoPQ is a two components system that positively regulates the accumulation of RpoS in S. Typhimurium. Under low magnesium concentrations, PhoP activates the transcription of IraP, a protein that prevents RpoS degradation by ClpXP. In our laboratory, we observed that RpoS accumulation is different between S. Typhi and S. Typhimurium, and we determined that RpoS accumulation in S. Typhi is increased in ĪphoPQ mutants. Previous bioinformatic analyses of S. Typhi and S. Typhimurium performed to try to explain these differences showed that our reference strain STH2370 lost a genomic segment where iraP was located. Thus, in this work, we incorporated iraP in S. Typhi STH2370 in a single copy and in its original genetic context, using a cis heterologous complementation technique and we evaluated its role in RpoS accumulation in the WT and ĪphoPQ strains, using lacZ fusions, Western blot analyses, protein stability assays and survival in hydrogen peroxide assays. In addition, we performed invasion and proliferation assays in HT-29 epithelial cells, to determine if the absence of iraP corresponds to a case of loss of function implicated in S. Typhi virulence. We found that IraP increases RpoS accumulation in low magnesium concentrations. Also, we found that IraP changes the regulation of RpoS mediated by PhoPQ. Finally, we observed that in an iraP cis complemented strain exhibited a decrease in the ability to invade HT-29 epithelial cells when compared to the WT strain. These results suggest that the loss of iraP in S. Typhi could involve certain evolutive advantages.
Salmonella Typhi (S. Typhi), the causative agent of typhoid fever, exclusively infects humans. Although S. Typhi is closely related to Salmonella Typhimurium (S. Typhimurium), they differ in host specificity. These differences are due to a multifactorial evolutive process that depends on gain functions, loss functions, and a differential regulation of the genes shared by both serovars. An example of the latter is the regulation of the alternative Sigma factor, RpoS, a master regulator of stress response, that contributes to the infective cycle of these pathogens. This Sigma factor is regulated at different levels, being proteolysis one of the most important regulation mechanisms. PhoPQ is a two components system that positively regulates the accumulation of RpoS in S. Typhimurium. Under low magnesium concentrations, PhoP activates the transcription of IraP, a protein that prevents RpoS degradation by ClpXP. In our laboratory, we observed that RpoS accumulation is different between S. Typhi and S. Typhimurium, and we determined that RpoS accumulation in S. Typhi is increased in ĪphoPQ mutants. Previous bioinformatic analyses of S. Typhi and S. Typhimurium performed to try to explain these differences showed that our reference strain STH2370 lost a genomic segment where iraP was located. Thus, in this work, we incorporated iraP in S. Typhi STH2370 in a single copy and in its original genetic context, using a cis heterologous complementation technique and we evaluated its role in RpoS accumulation in the WT and ĪphoPQ strains, using lacZ fusions, Western blot analyses, protein stability assays and survival in hydrogen peroxide assays. In addition, we performed invasion and proliferation assays in HT-29 epithelial cells, to determine if the absence of iraP corresponds to a case of loss of function implicated in S. Typhi virulence. We found that IraP increases RpoS accumulation in low magnesium concentrations. Also, we found that IraP changes the regulation of RpoS mediated by PhoPQ. Finally, we observed that in an iraP cis complemented strain exhibited a decrease in the ability to invade HT-29 epithelial cells when compared to the WT strain. These results suggest that the loss of iraP in S. Typhi could involve certain evolutive advantages.
Notas
Tesis (MagĆster en BiotecnologĆa)
Esta Tesis se realizĆ³ en el Laboratorio de GenĆ©tica y PatogĆ©nesis Bacteriana, Facultad de Ciencias BiolĆ³gicas, Universidad AndrĆ©s Bello y fue financiada por el Proyecto FONDECYT 11121506
Esta Tesis se realizĆ³ en el Laboratorio de GenĆ©tica y PatogĆ©nesis Bacteriana, Facultad de Ciencias BiolĆ³gicas, Universidad AndrĆ©s Bello y fue financiada por el Proyecto FONDECYT 11121506
Palabras clave
Salmonella Typhi, Enfermedades Infecciosas, Investigaciones