Rol de la proteína chaperona Hfq en la regulación de la virulencia del patógeno de salmónidos Yersinia Ruckeri
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Fecha
2022
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Idioma
es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
Yersinia ruckeri es una bacteria Gram negativa, agente causal de la enfermedad entérica de la boca
roja en salmónidos, enfermedad asociada a altas tasas de mortalidad, por lo que representa una
amenaza latente a la salud de los salmónidos y a la acuicultura, generando pérdidas económicas
significativas a países donde la actividad acuícola es relevante, como el caso de Chile.
La virulencia bacteriana, tanto de Y. ruckeri como otras bacterias, está asociada a sus diversos
procesos fisiológicos y respuestas a estrés, por lo cual es importante conocer los mecanismos
moleculares que la regulan, incluyendo aquellos que regulan la resistencia al estrés intra-hospedero y
que resultan fundamentales para la proliferación bacteriana en un proceso infectivo. Se reconoce que
los RNAs pequeños no codificantes (sRNAs) están involucrados en la regulación de varios procesos
fisiológicos y respuestas al estrés en las bacterias, mediante el apareamiento con mRNAs blancos y
la subsecuente regulación de su traducción. A su vez, muchos sRNAs requieren de la proteína
chaperona Hfq para cumplir sus funciones regulatorias sobre los mRNAs blancos. Es así como esta
proteína constituye un factor pleiotrópico en muchas bacterias, al estar involucrada en la regulación
de diversos procesos fisiológicos y respuestas al estrés.
El objetivo de este estudio fue evaluar el rol de la proteína Hfq en la modulación de respuestas a
condiciones de estrés intra-hospedero y virulencia de un aislado ambiental de Y. ruckeri, mediante
análisis fenotípicos y moleculares con una cepa carente del gen hfq, tanto en cultivos bacterianos
como en ensayos de infección in vitro.
Se evaluó la respuesta de una mutante de Y. ruckeri deficiente de Hfq, en comparación con una cepa
silvestre, frente a estrés oxidativo y estrés por déficit de hierro, con el propósito de aproximarse in
vitro a condiciones prevalecientes en el ambiente intra-hospedero. Se determinó que, bajo las
condiciones analizadas, la proteína Hfq no juega un rol crucial en la resistencia frente al estrés por
déficit de hierro, pero sí actúa como un modulador de la respuesta al estrés oxidativo evidenciándose
un mayor crecimiento en su ausencia. Por otra parte, se realizaron ensayos de infección in vitro en
una línea celular de salmón (CHSE-214), demostrando que la ausencia de la chaperona Hfq genera
un aumento en la proliferación y capacidad de infección.
Finalmente, se analizó la expresión de factores asociados a virulencia y de respuesta a estrés, bajo
condiciones de estrés oxidativo y por déficit de hierro, determinando que, en una cepa mutante con
deleción del gen hfq, la expresión aumentaba significativamente en la mayoría de los genes
estudiados.
En su conjunto, los resultados obtenidos en el presente estudio nos permiten evidenciar que la proteína
Hfq es requerida para modular respuestas asociadas a estrés y virulencia, reprimiendo la expresión de
diversos genes relacionados con estos procesos.
Yersinia ruckeri is a Gram-negative bacterium, causal agent of enteric redmouth disease in salmonids, a disease associated with high mortality rates, which represents a latent threat to the health of salmonids and aquaculture, generating significant economic losses to countries where aquaculture activity is relevant, such as Chile. Bacterial virulence, in Y. ruckeri and other bacteria, is associated with its diverse physiological processes and responses to stress, then it is important to know the molecular mechanisms that regulate it, including those that regulate resistance to intra-host stress which are fundamental for bacterial proliferation in an infective process. It is recognized that small non-coding RNAs (sRNAs) are involved in the regulation of several physiological processes and stress responses in bacteria, through pairing with target mRNAs and subsequent regulation of their translation. At the same time, many sRNAs require the chaperone protein Hfq to fulfill their regulatory functions on target mRNAs. Thus, this protein constitutes a pleiotropic factor in many bacteria, being involved in the regulation of diverse physiological processes and stress responses. The aim of this study was to evaluate the role of the Hfq protein in modulating responses to intra-host stress conditions and virulence of an environmental isolate of Y. ruckeri, through phenotypic and molecular analyses with a strain lacking the hfq gene, both in bacterial cultures and in vitro infection assays. The response of an Hfq-deficient Y. ruckeri mutant, in comparison with a wild-type strain, to oxidative stress and iron-deficiency stress was evaluated in order to resemble in vitro conditions prevailing in the intrahost environment. It was determined that, under the conditions analyzed, the Hfq protein does not play a crucial role in the resistance to iron deficiency stress, but it does act as a modulator of the response to oxidative stress, evidencing greater growth in its absence. On the other hand, in vitro infection assays were performed in a salmon cell line (CHSE-214), demonstrating that the absence of the Hfq chaperone generates an increase in proliferation and infection capacity. Finally, the expression of virulence and stress-associated factors was analyzed under conditions of oxidative stress and iron deficiency, determining that, in a mutant strain with deletion of the hfq gene, the expression of most of the genes studied was significantly increased. Taken together, the results obtained in the present study allow us to demonstrate that the Hfq protein is required to modulate responses associated with stress and virulence, repressing the expression of several genes related to these processes.
Yersinia ruckeri is a Gram-negative bacterium, causal agent of enteric redmouth disease in salmonids, a disease associated with high mortality rates, which represents a latent threat to the health of salmonids and aquaculture, generating significant economic losses to countries where aquaculture activity is relevant, such as Chile. Bacterial virulence, in Y. ruckeri and other bacteria, is associated with its diverse physiological processes and responses to stress, then it is important to know the molecular mechanisms that regulate it, including those that regulate resistance to intra-host stress which are fundamental for bacterial proliferation in an infective process. It is recognized that small non-coding RNAs (sRNAs) are involved in the regulation of several physiological processes and stress responses in bacteria, through pairing with target mRNAs and subsequent regulation of their translation. At the same time, many sRNAs require the chaperone protein Hfq to fulfill their regulatory functions on target mRNAs. Thus, this protein constitutes a pleiotropic factor in many bacteria, being involved in the regulation of diverse physiological processes and stress responses. The aim of this study was to evaluate the role of the Hfq protein in modulating responses to intra-host stress conditions and virulence of an environmental isolate of Y. ruckeri, through phenotypic and molecular analyses with a strain lacking the hfq gene, both in bacterial cultures and in vitro infection assays. The response of an Hfq-deficient Y. ruckeri mutant, in comparison with a wild-type strain, to oxidative stress and iron-deficiency stress was evaluated in order to resemble in vitro conditions prevailing in the intrahost environment. It was determined that, under the conditions analyzed, the Hfq protein does not play a crucial role in the resistance to iron deficiency stress, but it does act as a modulator of the response to oxidative stress, evidencing greater growth in its absence. On the other hand, in vitro infection assays were performed in a salmon cell line (CHSE-214), demonstrating that the absence of the Hfq chaperone generates an increase in proliferation and infection capacity. Finally, the expression of virulence and stress-associated factors was analyzed under conditions of oxidative stress and iron deficiency, determining that, in a mutant strain with deletion of the hfq gene, the expression of most of the genes studied was significantly increased. Taken together, the results obtained in the present study allow us to demonstrate that the Hfq protein is required to modulate responses associated with stress and virulence, repressing the expression of several genes related to these processes.
Notas
Tesis (Ingeniero en Biotecnología)
Palabras clave
Salmónidos, Enfermedades, Virulencia, Investigaciones, Proteínas, Análisis