Evaluación transcripcional de la respuesta inmune en Caligus rogercresseyi sobre salmón del Atlático inmunizados con vacuna antimicrobiota.
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Date
2024
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Publisher
Universidad de Concepción
Abstract
La comprensión de la respuesta inmune de los organismos ante la presencia de patógenos es crucial para el desarrollo de estrategias efectivas de control y prevención de enfermedades. En el contexto de la acuicultura, donde la salud de los peces es fundamental para el éxito de la industria, el estudio de los mecanismos inmunes de los parásitos que afectan a las poblaciones de peces es de suma importancia. En este sentido, Caligus rogercresseyi emerge como un agente patógeno de relevancia en la salmonicultura chilena, siendo una de las mayores problemáticas que afectan a las principales especies de salmónidos de exportación de nuestro país. Se sabe que el estado de salud equilibrado está estrechamente relacionado con la interacción entre el sistema inmunitario y la microbiota del organismo. La microbiota cumple funciones esenciales como el metabolismo de nutrientes y la resistencia a infestación de patógenos. Sin embargo cuando este equilibrio se ve alterado pueden generar una depresión del sistema inmune del organismo. Las vacunas antimicrobiota están diseñadas para intervenir este equilibrio, desfavoreciendo el microbioma del huesped y fortaleciendo el sistema del hospedador. En el caso específico de C. rogercresseyi, la interacción entre su microbiota y su sistema inmune aún no ha sido completamente explorada. Entender esta interacción es crucial, ya que puede proporcionar información valiosa sobre los mecanismos de defensa del parásito y potenciales puntos de intervención para el desarrollo de vacunas más efectiva. El objetivo del presente estudio fue evaluar la respuesta transcriptómica de los genes involucrados en vías de muerte celular o apoptosis, como también en la respuesta inmune innata, procesos de estrés oxidativo y secretoma en C. rogercresseyi expuesto a una vacuna antimicrobiota. Para ello se consideró un pool de 6 individuos de ambos sexos, extraídos de peces inmunizados con vacuna antimicrobiota formulada con un mix de bacterias inactivas aisladas desde C. rogercresseyi. La evaluación se realizó a los 25 y 68 días post-infección. Las muestras fueron secuenciadas con el objetivo de realizar un análisis in silico de expresión génica, y posteriormente se realizó una validación mediante qPCR. Como resultados, basado en el análisis bioinformático, se evidencia un considerable número de transcritos involucrados en vías metabólicas clave, como la apoptosis, la señalización de factores de necrosis tumoral (TNF) y Ferroptosis. De estas vías, la Ferroptosis fue la más consistentemente validada por los resultados de expresión relativa. Los análisis transcriptómicos basados en qPCR revelan una expresión diferencial de genes relacionados con homeostasis del hierro y la respuesta al estrés oxidativo, como ferritina, SOD, catalasa y PHGP. Además, el análisis in silico indica que la vacuna antimicrobiota induce una regulación diferencial de genes que codifican para ferroportina y ferritina, proteínas esenciales en la vía de la ferroptosis. Estas desempeñan roles críticos en la regulación del metabolismo del hierro y la homeostasis de las especies reactivas de oxígeno (ROS), y su sobreexpresión podría actuar como un mecanismo de defensa contra el estrés oxidativo, previniendo la muerte celular. Asimismo, los resultados de expresión diferencial muestran la sobreexpresión IKKβ, involucrado en la cascada de señalización de la respuesta inmune innata en C. rogercresseyi, lo que sugiere que la vacuna antimicrobiota parece modular parcialmente la respuesta inmune del ectoparásito frente al hospedador. Dentro de los resultados de expresión relativa no se evidencio un efecto negativo consistente en la capacidad del parásito para evadir la respuesta inmune del hospedador, ya que la modulación de genes como serpinas, catepsinas y tripsinas no mostró diferencias significativas con respecto a los estudios previos. En conclusión, los resultados de este estudio destacan el potencial de las vacunas antimicrobiotas para alterar vías de señalización críticas en C. rogercresseyi y contribuir a estrategias innovadoras para el control de este parásito en la salmonicultura, lo que puede minimizar los impactos negativos en el medio ambiente y mejorar la sostenibilidad de la producción.
Understanding the immune response of organisms in the presence of pathogens is crucial for the development of effective disease control and prevention strategies. In the context of aquaculture, where fish health is critical to the success of the industry, the study of the immune mechanisms of parasites affecting fish populations is of utmost importance. In this sense, Caligus rogercresseyi emerges as a pathogenic agent of relevance in Chilean salmon farming, being one of the major problems affecting the main species of salmonids exported from our country. It is known that a balanced state of health is closely related to the interaction between the immune system and the microbiota of the organism. The microbiota fulfills essential functions such as nutrient metabolism and resistance to pathogen infestation. However, when this balance is disturbed, it can lead to a depression of the body's immune system. Antimycobiota vaccines are designed to intervene in this balance, depleting the host microbiome and strengthening the host system. In the specific case of C. rogercresseyi, the interaction between its microbiota and its immune system has not yet been fully explored. Understanding this interaction is crucial, as it can provide valuable information on the defense mechanisms of the parasite and potential points of intervention for the development of more effective vaccines. The aim of the present study is to evaluate the transcriptomic response of genes involved in cell death pathways, as well as in the innate immune response, oxidative stress processes and secretomes in C. rogercresseyi exposed to an antimicrobiotic vaccine. For this purpose, a pool of 6 individuals of both sexes, extracted from fish immunized with vaccine formulated with a mix of inactivated bacteria, was considered after 25 and 68 dpi. The samples were sequenced in order to perform an in silico analysis of gene expression, and the results were subsequently validated by qPCR. As results, based on the bioinformatics analysis, a considerable number of transcripts involved in key metabolic pathways, such as apoptosis, tumor necrosis factor (TNF) signaling and Ferroptosis, are evident. Of these pathways, Ferroptosis was the most consistently validated by relative expression results. Transcriptomic analysis based on qPCR revealed differential expression of genes related to iron homeostasis and the response to oxidative stress, such as ferritin, SOD, catalase and PHGP. Furthermore, in silico analysis indicates that the vaccine induces differential regulation of genes encoding for ferroportin and ferritin, essential proteins in the ferroptosis pathway. These play critical roles in the regulation of iron metabolism and reactive oxygen species (ROS) homeostasis, and their overexpression could act as a defense mechanism against oxidative stress, preventing cell death. Likewise, the differential expression results show the overexpression of IKKβ, involved in the signaling cascade of the innate immune response in C. rogercresseyi, suggesting that the antimicrobiotic vaccine appears to partially modulate the immune response of the ectoparasite against the host. Within the results of relative expression, there was no evidence of a consistent negative effect on the parasite's ability to evade the host's immune response of the host, since the modulation of genes such as serpins, cathepsins and trypsins did not show significant differences with respect to previous studies. In conclusion, the results of this study highlight the potential of antimicrobial vaccines to alter critical signaling pathways in C. rogercressey and to contribute to innovative strategies for the control of this parasite in salmon farming, which can minimize negative impacts on the environment and improve production sustainability.
Understanding the immune response of organisms in the presence of pathogens is crucial for the development of effective disease control and prevention strategies. In the context of aquaculture, where fish health is critical to the success of the industry, the study of the immune mechanisms of parasites affecting fish populations is of utmost importance. In this sense, Caligus rogercresseyi emerges as a pathogenic agent of relevance in Chilean salmon farming, being one of the major problems affecting the main species of salmonids exported from our country. It is known that a balanced state of health is closely related to the interaction between the immune system and the microbiota of the organism. The microbiota fulfills essential functions such as nutrient metabolism and resistance to pathogen infestation. However, when this balance is disturbed, it can lead to a depression of the body's immune system. Antimycobiota vaccines are designed to intervene in this balance, depleting the host microbiome and strengthening the host system. In the specific case of C. rogercresseyi, the interaction between its microbiota and its immune system has not yet been fully explored. Understanding this interaction is crucial, as it can provide valuable information on the defense mechanisms of the parasite and potential points of intervention for the development of more effective vaccines. The aim of the present study is to evaluate the transcriptomic response of genes involved in cell death pathways, as well as in the innate immune response, oxidative stress processes and secretomes in C. rogercresseyi exposed to an antimicrobiotic vaccine. For this purpose, a pool of 6 individuals of both sexes, extracted from fish immunized with vaccine formulated with a mix of inactivated bacteria, was considered after 25 and 68 dpi. The samples were sequenced in order to perform an in silico analysis of gene expression, and the results were subsequently validated by qPCR. As results, based on the bioinformatics analysis, a considerable number of transcripts involved in key metabolic pathways, such as apoptosis, tumor necrosis factor (TNF) signaling and Ferroptosis, are evident. Of these pathways, Ferroptosis was the most consistently validated by relative expression results. Transcriptomic analysis based on qPCR revealed differential expression of genes related to iron homeostasis and the response to oxidative stress, such as ferritin, SOD, catalase and PHGP. Furthermore, in silico analysis indicates that the vaccine induces differential regulation of genes encoding for ferroportin and ferritin, essential proteins in the ferroptosis pathway. These play critical roles in the regulation of iron metabolism and reactive oxygen species (ROS) homeostasis, and their overexpression could act as a defense mechanism against oxidative stress, preventing cell death. Likewise, the differential expression results show the overexpression of IKKβ, involved in the signaling cascade of the innate immune response in C. rogercresseyi, suggesting that the antimicrobiotic vaccine appears to partially modulate the immune response of the ectoparasite against the host. Within the results of relative expression, there was no evidence of a consistent negative effect on the parasite's ability to evade the host's immune response of the host, since the modulation of genes such as serpins, cathepsins and trypsins did not show significant differences with respect to previous studies. In conclusion, the results of this study highlight the potential of antimicrobial vaccines to alter critical signaling pathways in C. rogercressey and to contribute to innovative strategies for the control of this parasite in salmon farming, which can minimize negative impacts on the environment and improve production sustainability.
Description
Tesis presentada para optar al título de Biólogo Marino
Keywords
Salmón del atlántico, Peces Investigaciones, Vacunas