Efecto de la alta osmolaridad en la entrega de factores de virulencia a células epiteliales por OMVs (Outer Membrane Vesicles) de Salmonella enterica serovar Typhi
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2023
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es
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Universidad Andrés Bello
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Licencia CC
Resumen
Salmonella enterica serovar Typhi (S. Typhi) es un patógeno Gram negativo
intracelular estricto de humanos y es el agente etiológico de la fiebre tifoidea. La invasión de
Salmonella en células intestinales está mediada por el Sistema de Secreción de Tipo III
(TTSSIII) y su expresión es regulada por señales ambientales presentes en el intestino, como
la alta osmolaridad. La alta osmolaridad promueve la expresión de proteínas asociadas al
TTSSIII, como SipB y SipC, que conforman el translocón del TTSSIII. El translocón se
inserta en la membrana de la célula hospedera y forma un poro para permitir la inyección de
proteínas que favorecen la internalización de S. Typhi. Existe evidencia genética que indica
que SipB y SipC son secretados por el inyectisoma hacia el medio extracelular, y de esta
manera llegan a la membrana de la célula hospedera para formar el translocón del TTSSIII,
sin embargo, creemos que pueden existir mecanismos alternativos de entrega de estos
factores de virulencia.
Las vesículas de membrana externa (OMVs, del inglés Outer Membrane Vesicles)
son proteoliposomas con estructuras heterogéneas del orden de los 20 a 200 nm, liberadas
desde la membrana externa de bacterias Gram negativo. Las OMVs son capaces de
transportar biomoléculas y destacan por su capacidad de entrega de factores de virulencia.
Además, se ha reportado que las propiedades y el cargamento de OMVs pueden ser
modificadas según las condiciones de cultivo. Experimentos preliminares indican que las
OMVs provenientes de bacterias de S. Typhi en condiciones intestinales son capaces de ser
internalizadas a mayor tasa y en un mayor porcentaje de células en cultivo, en comparación
con OMVs provenientes de S. Typhi en condiciones estándar, sugiriendo un cambio en las
propiedades de las OMVs dado por las condiciones de cultivo. Hipotetizamos que la alta
osmolaridad cambia propiedades de OMVs de S. Typhi, favorece la internalización OMVs y entrega de factores de virulencia en células epiteliales a través de OMVs. Se compararon
OMVs obtenidas desde condiciones de cultivo estándar y condiciones de cultivo con alta
osmolaridad. El perfil proteico de OMVs se estudió por SDS-PAGE. La presencia de factores
de virulencia en las OMVs se determinó por Western Blot. La internalización de OMVs se
analizó por citometría de flujo y la entrega de factores de virulencia a células epiteliales se
determinó por inmunocitoquímica. También estudiamos entrega de factores de virulencia
utilizando actividad β-lactamasa como reportero. Encontramos que la alta osmolaridad
modula el cargamento de proteínas en OMVs de S. Typhi. La alta osmolaridad aumenta la
carga de SipC en OMVs de S. Typhi. Además, la alta osmolaridad favorece la internalización
de OMVs en células epiteliales y también favorece la entrega de SipC a células epiteliales
mediante OMVs. Como conclusión, encontramos que la alta osmolaridad cambia el perfil de
proteínas OMVs, aumenta la carga de factores de virulencia y entrega de estos en células
epiteliales a través de OMVs. Estos hallazgos pueden ayudar a dilucidar la potencial
participación de OMVs de S. Typhi en su ciclo infectivo.
Salmonella enterica serovar Typhi (S. Typhi) is a strict intracellular Gram-negative pathogen of humans and is the etiological agent of typhoid fever. The invasion of Salmonella into intestinal cells is mediated by the Type III Secretion System (TTSSIII), and its expression is regulated by environmental signals present in the intestine, such as high osmolarity. High osmolarity promotes the expression of TTSSIII-associated proteins, such as SipB and SipC, which make up the TTSSIII translocon. The translocon inserts into the host cell membrane and forms a pore to allow the injection of proteins that promote S. Typhi's internalization. Genetic evidence shows that the injectisome secretes SipB and SipC into the extracellular milieu before reaching the host cell membrane to form the TTSSIII translocon; however, we believe that there may be alternative delivery mechanisms for these virulence factors. Outer membrane vesicles (OMVs) are proteoliposomes with heterogeneous structures of 20 to 200 nm, released from the outer membrane of Gram-negative bacteria. OMVs can transport biomolecules and are noted for their ability to deliver virulence factors. In addition, it has been reported that the OMV properties and cargo can be modified depending on the culture conditions. Preliminary experiments indicate that OMVs from S. Typhi bacteria under intestinal conditions can be internalized at a higher rate and in a higher percentage of cells in culture compared to OMVs from S. Typhi under standard conditions, suggesting a change in OMV properties given by culture conditions. We hypothesized that high osmolarity changes the properties of OMVs from S. Typhi, favoring OMV internalization and delivery of virulence factors into epithelial cells through OMVs. OMVs obtained from standard culture conditions, and high osmolarity culture conditions were compared. The protein profile of OMVs was studied by SDS-PAGE. The presence of virulence factors in OMVs was determined by western blot. Internalization of OMVs was analyzed by flow cytometry, and delivery of virulence factors to epithelial cells was determined by immunocytochemistry. We also studied virulence factor delivery using β-lactamase activity as a reporter. We found that high osmolarity modulates protein loading in S. Typhi OMVs. In addition, high osmolarity increases SipC loading in S. Typhi OMVs. Furthermore, high osmolarity favors the internalization of OMVs into epithelial cells and also favors the delivery of SipC to epithelial cells by OMVs. In conclusion, we found that high osmolarity changes the OMVs protein profile and increases virulence factor loading and delivery of virulence factors into epithelial cells via OMVs. These findings may help to elucidate the potential involvement of OMVs of S. Typhi in its infectious cycle.
Salmonella enterica serovar Typhi (S. Typhi) is a strict intracellular Gram-negative pathogen of humans and is the etiological agent of typhoid fever. The invasion of Salmonella into intestinal cells is mediated by the Type III Secretion System (TTSSIII), and its expression is regulated by environmental signals present in the intestine, such as high osmolarity. High osmolarity promotes the expression of TTSSIII-associated proteins, such as SipB and SipC, which make up the TTSSIII translocon. The translocon inserts into the host cell membrane and forms a pore to allow the injection of proteins that promote S. Typhi's internalization. Genetic evidence shows that the injectisome secretes SipB and SipC into the extracellular milieu before reaching the host cell membrane to form the TTSSIII translocon; however, we believe that there may be alternative delivery mechanisms for these virulence factors. Outer membrane vesicles (OMVs) are proteoliposomes with heterogeneous structures of 20 to 200 nm, released from the outer membrane of Gram-negative bacteria. OMVs can transport biomolecules and are noted for their ability to deliver virulence factors. In addition, it has been reported that the OMV properties and cargo can be modified depending on the culture conditions. Preliminary experiments indicate that OMVs from S. Typhi bacteria under intestinal conditions can be internalized at a higher rate and in a higher percentage of cells in culture compared to OMVs from S. Typhi under standard conditions, suggesting a change in OMV properties given by culture conditions. We hypothesized that high osmolarity changes the properties of OMVs from S. Typhi, favoring OMV internalization and delivery of virulence factors into epithelial cells through OMVs. OMVs obtained from standard culture conditions, and high osmolarity culture conditions were compared. The protein profile of OMVs was studied by SDS-PAGE. The presence of virulence factors in OMVs was determined by western blot. Internalization of OMVs was analyzed by flow cytometry, and delivery of virulence factors to epithelial cells was determined by immunocytochemistry. We also studied virulence factor delivery using β-lactamase activity as a reporter. We found that high osmolarity modulates protein loading in S. Typhi OMVs. In addition, high osmolarity increases SipC loading in S. Typhi OMVs. Furthermore, high osmolarity favors the internalization of OMVs into epithelial cells and also favors the delivery of SipC to epithelial cells by OMVs. In conclusion, we found that high osmolarity changes the OMVs protein profile and increases virulence factor loading and delivery of virulence factors into epithelial cells via OMVs. These findings may help to elucidate the potential involvement of OMVs of S. Typhi in its infectious cycle.
Notas
Tesis (Magíster en Biotecnología y Ciencias de la Vida)
Tesis financiada por FONDECYT REGULAR 1220584.
Tesis financiada por FONDECYT REGULAR 1220584.
Palabras clave
Concentración Osmolar, Células Epiteliales, Salmonella Typhi, Salmonella Enteritidis