Internalización de proteínas efectoras SipB y SipC mediante vesículas de membrana externa de Salmonella Typhi en células epiteliales
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Fecha
2023
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es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
Salmonella entérica serovar Typhi (S. Typhi) es el causante etiológico de la fiebre
tifoidea, teniendo como hospedero único al ser humano. Dentro de los
mecanismos que utiliza Salmonella para infectar y entregar factores de virulencia
se encuentra el sistema de secreción tipo 3 (T3SS), el que se une a la célula
hospedera mediante la acción de las proteínas SipB y SipC. Estas proteínas
conforman el translocón (poro), que se sitúa en la membrana de la célula
hospedera, permitiendo una conexión directa entre el citoplasma bacteriano y la
célula diana.
Por su parte, las vesículas de membrana externa (OMVs), son proteoliposomas
nanométricos que se desprenden naturalmente de las bacterias, desempeñando
diversas funciones. Pese a que, en los últimos años, estas vesículas han ido
ganando atención, la funcionalidad de las OMVs de S. Typhi es desconocida.
Estudios de proteómica realizados en nuestro laboratorio indican que SipB y SipC
se encuentran presentes en OMVs de S. Typhi. Además, observamos que las
OMVs de S. Typhi son internalizadas por células epiteliales. En base a lo ya
descrito y con la finalidad de comprender más a detalle si las OMVs de S. Typhi
realizan la entrega de proteínas, proponemos como hipótesis, las proteínas del
translocón SipB y SipC pueden ser entregadas a células epiteliales mediante
OMVs de Salmonella Typhi.
Utilizando la técnica de Gibson Assembly se fusionaron las proteínas SipB y
SipC con el reportero TEM-1 (β-Lactamasa) en un vector y se insertaron en una
cepa silvestre (WT) de S. Typhi. Para dilucidar la entrega de sipB-TEM y sipCTEM se utilizó el ensayo de FRET utilizando como reportero la β-lactamasa. Se
observó que las células con sipB-TEM y sipC-TEM presentan fluorescencia
asociada a translocación a las células epiteliales por lo que es plausible decir que
las OMVs de S. Typhi actúan como vehículos de entrega de efectores del T3SS1 asociados al translocón. A futuro las metodologías aplicadas en este trabajo
pueden extrapolarse al estudio de otros factores de virulencia y comprender
cuales de estos poseen la capacidad de ser entregados mediante OMVs.
Salmonella enterica serovar Typhi (S. Typhi) is the etiological cause of typhoid fever, with humans as its sole host. Among the mechanisms that Salmonella uses to infect and deliver virulence factors is the type 3 secretion system (T3SS), which binds to the host cell through the action of the SipB and SipC proteins. These proteins make up the translocon (pore), which is located in the membrane of the host cell, allowing a direct connection between the bacterial cytoplasm and the target cell. For their part, outer membrane vesicles (OMVs) are nanometric proteoliposomes that are naturally released from bacteria, performing various functions. Although these vesicles have been gaining attention in recent years, the functionality of S. Typhi OMVs is unknown. Proteomics studies carried out in our laboratory indicate that SipB and SipC are present in S. Typhi OMVs. Furthermore, we observed that S. Typhi OMVs are internalized by epithelial cells. Based on what has already been described and in order to understand in more detail whether S. Typhi OMVs deliver proteins, we propose as a hypothesis that the SipB and SipC translocon proteins can be delivered to epithelial cells by Salmonella Typhi OMVs. Using the Gibson Assembly technique, the SipB and SipC proteins were fused with the TEM-1 (β-lactamase) reporter in a vector and inserted into a wild-type (WT) S. Typhi strain. To elucidate the delivery of sipB-TEM and sipC-TEM, the FRET assay was used using β-lactamase as a reporter. It was observed that cells with sipB-TEM and sipC-TEM present fluorescence associated with translocation to epithelial cells, so it is plausible to say that S. Typhi OMVs act as delivery vehicles for T3SS-1 effectors associated with the translocon. In the future, the methodologies applied in this work can be extrapolated to the study of other virulence factors and understand which of these have the capacity to be delivered through OMVs.
Salmonella enterica serovar Typhi (S. Typhi) is the etiological cause of typhoid fever, with humans as its sole host. Among the mechanisms that Salmonella uses to infect and deliver virulence factors is the type 3 secretion system (T3SS), which binds to the host cell through the action of the SipB and SipC proteins. These proteins make up the translocon (pore), which is located in the membrane of the host cell, allowing a direct connection between the bacterial cytoplasm and the target cell. For their part, outer membrane vesicles (OMVs) are nanometric proteoliposomes that are naturally released from bacteria, performing various functions. Although these vesicles have been gaining attention in recent years, the functionality of S. Typhi OMVs is unknown. Proteomics studies carried out in our laboratory indicate that SipB and SipC are present in S. Typhi OMVs. Furthermore, we observed that S. Typhi OMVs are internalized by epithelial cells. Based on what has already been described and in order to understand in more detail whether S. Typhi OMVs deliver proteins, we propose as a hypothesis that the SipB and SipC translocon proteins can be delivered to epithelial cells by Salmonella Typhi OMVs. Using the Gibson Assembly technique, the SipB and SipC proteins were fused with the TEM-1 (β-lactamase) reporter in a vector and inserted into a wild-type (WT) S. Typhi strain. To elucidate the delivery of sipB-TEM and sipC-TEM, the FRET assay was used using β-lactamase as a reporter. It was observed that cells with sipB-TEM and sipC-TEM present fluorescence associated with translocation to epithelial cells, so it is plausible to say that S. Typhi OMVs act as delivery vehicles for T3SS-1 effectors associated with the translocon. In the future, the methodologies applied in this work can be extrapolated to the study of other virulence factors and understand which of these have the capacity to be delivered through OMVs.
Notas
Tesis (Licenciado en Biología)
Proyectos del laboratorio FONDECYT 1220584 y FONDECYT 1181638.
Proyectos del laboratorio FONDECYT 1220584 y FONDECYT 1181638.
Palabras clave
Proteínas, Salmonella Typhi, Células Epiteliales, Membrana Celular