Regulación de genes implicados en la virulencia de Brucella abortus mediante el sistema de transducción de señales de dos componentes BVRR/BVRS
Fecha
2023-12-13
Tipo
tesis doctoral
Autores
Rivas Solano, Olga Lilliana
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Resumen
B. abortus es un patógeno zoonótico intracelular-extracelular facultativo. Su virulencia se relaciona con su capacidad para invadir y replicarse dentro de células hospederas. Estudios previos con mutantes por transposición en los genes bvrR y bvrS que constituyen el sistema de dos componentes BvrR/BvrS, han demostrado que estas cepas son atenuadas y que BvrR/BvrS regula más de 100 genes relevantes para la virulencia. Sin embargo, no se han reportado mutantes por deleción para comparar fenotipos, y confirmar que la atenuación observada se debe directamente a la ausencia de BvrR/BvrS. No se conoce si BvrR/BvrS regula todos esos genes de forma directa, pues algunos de ellos codifican factores de transcripción. Entre los pocos genes cuya expresión se ha demostrado que está regulada positivamente por BvrR/BvrS, destacan: omp25, que codifica para una proteína de membrana externa conservada en Brucella con funciones estructurales e inmunomoduladoras; y el circuito de virulencia BvrR-VjbR-VirB, que es necesario para el tránsito intracelular, la replicación bacteriana, la salida de la célula hospedera y la infección de nuevas células. Además, en Brucella, pocas regiones promotoras han sido estudiadas. En esta tesis, construimos una cepa mutante de B. abortus con una doble deleción en bvrR/bvrS y demostramos que presenta un fenotipo atenuado similar al de las mutantes por transposición, lo que contribuye a validar el papel de BvrR/BvrS en la virulencia. También demostramos bvrR y bvrS pertenecen a un operón de 16 genes cuya organización transcripcional se encuentra conservada solo en miembros del orden Rhizobiales capaces de transitar entre ambientes extra e intracelulares, lo que respalda la importancia de este operón para la virulencia. Además, estudiamos el regulón de BvrR en condiciones que simulan el ambiente intracelular e identificamos regiones genómicas que se pueden unir a BvrR y que están asociadas a genes diana posiblemente regulados de forma directa por BvrR y con funciones relevantes para la virulencia. Confirmamos unión directa de BvrR corriente arriba los siguientes genes relacionados con la virulencia: omp25, tamA, pckA y bvrR, lo que implica autoregulación del operón bvrR/bvrS. Reportamos un sitio de unión a BvrR en el promotor del operón virB, el cual también es regulado por otros factores de transcripción adicionales. Contrario a lo reportado en Sinorhizobium meliloti con ortólogos de BvrR y del regulador transcripcional TetR2, no encontramos interacción directa entre BvrR y tetR2, el cual regula la expresión de vjbR en B. abortus en conjunto con otros factores de transcripción, incluyendo BvrR, lo que podría indicar un evento evolutivo relacionado con virulencia, pues S. meliloti es un endosimbionte. Además, caracterizamos la región reguladora de omp25 como prototipo de región regulada por BvrR y encontramos que presenta tres sitios de unión a BvrR y dos sitios de inicio de la transcripción, lo que sugiere una regulación diferencial en respuesta a condiciones ambientales. En conclusión, nuestros resultados contribuyen a comprender mejor la regulación génica de la virulencia a través de BvrR/BvrS en B. abortus.
B. abortus is an intracellular extracellular zoonotic pathogen. Its virulence depends on its ability to invade host cells and replicate within them. Previous studies with two transposition mutants in the genes bvrR and bvrS, both encoding the Two-Component System BvrR/BvrS, revealed attenuation of the mutant strains, and a role of BvrR/BvrS in the regulation of more than 100 virulence-related genes. However, deletion mutants have not been constructed to compare phenotypes, and to confirm that the attenuation is a consequence of the lack of BvrR/BvrS. It is unknown if all these target genes are directly regulated by BvrR/BvrS, because some of them encode other transcriptional regulators. The gene omp25, is among the few known positively regulated target genes. It encodes an outer membrane protein conserved in Brucella and it has structural and immunomodulatory roles. Other known target genes are vjbR and virB. The virulence circuit BvrR-VjbR-VirB is necessary for intracellular trafficking, bacterial replication, cell egress and infection of new cells. Moreover, in Brucella, few promoter regions have been studied. In this work, we constructed a B. abortus mutant strain with a double deletion in the genes bvrR and bvrS, which is attenuated like both transposition mutants, contributing to establish the role of BvrR/BvrS in virulence. We also demonstrated that bvrR and bvrS belong to an operon of 16 genes whose transcriptional organization is conserved in Rhizobiales members capable of transiting between extracellular and intracellular environments, which reinforces the relevance of this operon for virulence. Additionally, we studied the regulon of BvrR under conditions mimicking the intracellular environment and we identified genomic regions bound to BvrR and associated to virulence-related target genes possibly regulated directly by BvrR. We confirmed the direct binding of BvrR to the upstream region of the virulence-related genes: omp25, tamA, pckA and bvrR, implying autoregulation of the bvrR/bvrS operon. We also reported a BvrR binding site on the promoter of the virB operon. This operon is also regulated by other transcription factors. Contrary to what has been reported for Sinorhizobium meliloti, the transcriptional regulator TetR2 does not interact directly with BvrR. TetR2 regulates vjbR expression in B. abortus along with other transcription factors, besides BvrR. This could be related to a virulence-related evolutive event because S. meliloti is an endosymbiont. Moreover, we also characterized the regulatory region of omp25, as a prototype of a region directly regulated by BvrR, and we found three BvrR binding sites within it, suggesting differential regulation in response to environmental conditions. In conclusion, our results contribute to a better understanding of the gene regulation of virulence through BvrR/BvrS in B. abortus.
B. abortus is an intracellular extracellular zoonotic pathogen. Its virulence depends on its ability to invade host cells and replicate within them. Previous studies with two transposition mutants in the genes bvrR and bvrS, both encoding the Two-Component System BvrR/BvrS, revealed attenuation of the mutant strains, and a role of BvrR/BvrS in the regulation of more than 100 virulence-related genes. However, deletion mutants have not been constructed to compare phenotypes, and to confirm that the attenuation is a consequence of the lack of BvrR/BvrS. It is unknown if all these target genes are directly regulated by BvrR/BvrS, because some of them encode other transcriptional regulators. The gene omp25, is among the few known positively regulated target genes. It encodes an outer membrane protein conserved in Brucella and it has structural and immunomodulatory roles. Other known target genes are vjbR and virB. The virulence circuit BvrR-VjbR-VirB is necessary for intracellular trafficking, bacterial replication, cell egress and infection of new cells. Moreover, in Brucella, few promoter regions have been studied. In this work, we constructed a B. abortus mutant strain with a double deletion in the genes bvrR and bvrS, which is attenuated like both transposition mutants, contributing to establish the role of BvrR/BvrS in virulence. We also demonstrated that bvrR and bvrS belong to an operon of 16 genes whose transcriptional organization is conserved in Rhizobiales members capable of transiting between extracellular and intracellular environments, which reinforces the relevance of this operon for virulence. Additionally, we studied the regulon of BvrR under conditions mimicking the intracellular environment and we identified genomic regions bound to BvrR and associated to virulence-related target genes possibly regulated directly by BvrR. We confirmed the direct binding of BvrR to the upstream region of the virulence-related genes: omp25, tamA, pckA and bvrR, implying autoregulation of the bvrR/bvrS operon. We also reported a BvrR binding site on the promoter of the virB operon. This operon is also regulated by other transcription factors. Contrary to what has been reported for Sinorhizobium meliloti, the transcriptional regulator TetR2 does not interact directly with BvrR. TetR2 regulates vjbR expression in B. abortus along with other transcription factors, besides BvrR. This could be related to a virulence-related evolutive event because S. meliloti is an endosymbiont. Moreover, we also characterized the regulatory region of omp25, as a prototype of a region directly regulated by BvrR, and we found three BvrR binding sites within it, suggesting differential regulation in response to environmental conditions. In conclusion, our results contribute to a better understanding of the gene regulation of virulence through BvrR/BvrS in B. abortus.
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BACTERIA, GEN, GENÉTICA, PATOLOGÍA, ENFERMEDAD ANIMAL, MICROBIOLOGÍA