Evaluación de la expresión de la enzima ADP-ribosiltransferasa NarE durante la invasión de epitelio de trompa de Falopio humana con Neisseria gonorrhoeae
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2018
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es
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Universidad Andrés Bello
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Neisseria gonorrhoeae (gonococo) es una bacteria Gram-negativo, patógeno exclusivo del ser humano y agente etiológico de la infección de transmisión sexual gonorrea. La incidencia de esta infección en la población ha ido en aumento desde el año 2009 y si bien la infección posee tratamiento, la multirresistencia a antimicrobianos que posee gonococo y las secuelas crónicas que puede dejar en la mujer son de gran importancia, esto debido a que si la infección asciende hasta las trompas de Falopio existe el riesgo de infertilidad. Se ha reportado que N. gonorrhoeae es capaz de invadir las células del epitelio tubárico mediante el reordenamiento del citoesqueleto, sin embargo, se desconocen las bases moleculares de esta remodelación. En nuestro laboratorio se demostró que gonococo posee el gen narE previamente identificado en Neisseria meningitidis, el cual codifica para la toxina NarE, una ADP-ribosiltransferasa. Se ha reportado que en otros patógenos bacterianos estas toxinas participan en la invasión mediante la modificación de proteínas del citoesqueleto de la célula blanco por una ADP-ribosilación. El principal objetivo de esta Tesis es evaluar la expresión del gen narE de Neisseria gonorrhoeae y detectar la presencia de la enzima durante la infección de células epiteliales de trompas de Falopio humana. Para ello se realizó una extracción de ARN bacteriano antes de infectar cultivos primarios y luego a distintos tiempos posteriores a la infección (4, 8, 12 y 24 horas) y se evaluó la expresión del gen narE mediante RT-qPCR. Posteriormente se realizó una fusión génica de narE con el epítope 3xFLAG para la detección de NarE-3xFLAG mediante Western blot en las condiciones antes descritas. Nuestros resultados mostraron que en N. gonorrhoeae hubo una expresión
constitutiva del gen narE y que cuando el patógeno se encontró en contacto con células epiteliales de trompa de Falopio humana la expresión del gen narE aumentó durante las primeras horas de la infección (4 horas post-infección), mientras que la expresión disminuyó en los últimos tiempos de infección (12 y 24 horas post-infección). Además, se detectó la enzima en la fracción periplasmática de la bacteria. Posteriormente la proteína NarE fue detectada en los tiempos de 4, 8, 12 y 24 horas post infección. Adicionalmente se detectó que NarE apareció en una fracción de proteínas asociadas al citoesqueleto (E-cadherina, β-tubulina). Estos resultados sugieren que la enzima NarE de Neisseria gonorrhoeae participa en la modificación del citoesqueleto de células epiteliales de trompas de Falopio humana y que este efecto es importante en las primeras horas de infección. Por ello, es probable que NarE sea parte de los mecanismos moleculares utilizados por la bacteria durante el proceso de invasión de células epiteliales de trompas de Falopio humanas.
Neisseria gonorrhoeae (gonococcus) is a Gram-negative, human exclusive pathogen and the etiologic agent of the sexually-transmitted infection named gonorrhea. Although gonorrhea is not lethal, the high multidrug resistance displayed by gonococcus and the long-term sequelae in women are critical topics of study in gonococcal pathogenesis, since the infection can reach the fallopian tubes and thus a risk of cause infertility is possible. It has been reported that N. gonorrhoeae can invade the tubal epithelial cells through host cytoskeleton remodeling, however, the molecular basis of this remodeling is unknown. Our laboratory demonstrated that gonococcus possesses the narE gene previously identified in Neisseria meningitidis, which encodes the NarE toxin, a protein with ADP-ribosyltransferase activity. In other bacterial pathogens, this type of toxin participates in host cell invasion through modification of host cytoskeletal proteins by an ADP-ribosylation. The aim of this work is to evaluate the expression of the narE gene from Neisseria gonorrhoeae during the infection of human fallopian tube epithelial cells at 4, 8, 12 and 24-hour post-infection and detect the presence of the enzyme by Western blot. Bacterial RNA extraction was carried out before infection and then at 4, 8, 12 and 24-hours post-infection in primary cultures of human fallopian tubes epithelial cells. The expression of narE gene was assessed by RT-qPCR. Then a translational fusion with the 3xFLAG epitope was done and the fusion protein was detected by Western Blot in all the conditions previously described. Finally, purification of total and host cytoskeleton-associated proteins after infection was carried out and detection of NarE-3xFLAG was done by Western Blot. Our data showed that the gonococcal narE gene was constitutively expressed, which increased during the first hours of infection of human Fallopian tube epithelial cells (4-hour post-infection), but decreased at 12 and 24-hour post-infection. The NarE protein was only detected in the periplasmic fraction of N. gonorrhoeae, but the corresponding protein band appeared during infection assays of fallopian tubes epithelial cells at all the times of infection used in this study. Finally, NarE-3xFLAG was also found in the host cytoskeleton associated proteins fraction. These results suggest that the protein NarE of Neisseria gonorrhoeae may play a role in host cytoskeletal remodeling when invades the human Fallopian tube epithelia and this effect might be important during the early stage of infection. Additionally, our results suggest that NarE of Neisseria gonorrhoeae might be part of the molecular mechanisms used by the pathogen during the invasion process of human fallopian tube epithelial cells.
Neisseria gonorrhoeae (gonococcus) is a Gram-negative, human exclusive pathogen and the etiologic agent of the sexually-transmitted infection named gonorrhea. Although gonorrhea is not lethal, the high multidrug resistance displayed by gonococcus and the long-term sequelae in women are critical topics of study in gonococcal pathogenesis, since the infection can reach the fallopian tubes and thus a risk of cause infertility is possible. It has been reported that N. gonorrhoeae can invade the tubal epithelial cells through host cytoskeleton remodeling, however, the molecular basis of this remodeling is unknown. Our laboratory demonstrated that gonococcus possesses the narE gene previously identified in Neisseria meningitidis, which encodes the NarE toxin, a protein with ADP-ribosyltransferase activity. In other bacterial pathogens, this type of toxin participates in host cell invasion through modification of host cytoskeletal proteins by an ADP-ribosylation. The aim of this work is to evaluate the expression of the narE gene from Neisseria gonorrhoeae during the infection of human fallopian tube epithelial cells at 4, 8, 12 and 24-hour post-infection and detect the presence of the enzyme by Western blot. Bacterial RNA extraction was carried out before infection and then at 4, 8, 12 and 24-hours post-infection in primary cultures of human fallopian tubes epithelial cells. The expression of narE gene was assessed by RT-qPCR. Then a translational fusion with the 3xFLAG epitope was done and the fusion protein was detected by Western Blot in all the conditions previously described. Finally, purification of total and host cytoskeleton-associated proteins after infection was carried out and detection of NarE-3xFLAG was done by Western Blot. Our data showed that the gonococcal narE gene was constitutively expressed, which increased during the first hours of infection of human Fallopian tube epithelial cells (4-hour post-infection), but decreased at 12 and 24-hour post-infection. The NarE protein was only detected in the periplasmic fraction of N. gonorrhoeae, but the corresponding protein band appeared during infection assays of fallopian tubes epithelial cells at all the times of infection used in this study. Finally, NarE-3xFLAG was also found in the host cytoskeleton associated proteins fraction. These results suggest that the protein NarE of Neisseria gonorrhoeae may play a role in host cytoskeletal remodeling when invades the human Fallopian tube epithelia and this effect might be important during the early stage of infection. Additionally, our results suggest that NarE of Neisseria gonorrhoeae might be part of the molecular mechanisms used by the pathogen during the invasion process of human fallopian tube epithelial cells.
Notas
Tesis (Magíster en Biotecnología)
La presente Tesis se realizó en el Center for Integrative Medicine and Innovative Science (CIMIS) de la Facultad de Medicina de la Universidad Andrés Bello y fue financiada por el proyecto FONDECYT INICIACIÓN 11121262 titulado “Role of the ADP- ribosyltransferase toxin NarE in actin cytoskeleton remodeling during the invasion of the human Fallopian tube epithelium by Neisseria gonorrhoeae” y el proyecto UNAB DI-273-13/R.
La presente Tesis se realizó en el Center for Integrative Medicine and Innovative Science (CIMIS) de la Facultad de Medicina de la Universidad Andrés Bello y fue financiada por el proyecto FONDECYT INICIACIÓN 11121262 titulado “Role of the ADP- ribosyltransferase toxin NarE in actin cytoskeleton remodeling during the invasion of the human Fallopian tube epithelium by Neisseria gonorrhoeae” y el proyecto UNAB DI-273-13/R.
Palabras clave
Gonorrea, Trompas de Falopio, Infecciones