Role of the R. inulinivorans flagellum in adherence to intestinal constituents and gut colonization
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Archivos
Fecha
2023
Autores
Profesor/a Guía
Facultad/escuela
Idioma
es
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Universidad Andrés Bello
Nombre de Curso
Licencia CC
Licencia CC
Resumen
La microbiota intestinal (MI) humana, benéfica para la salud del ser humano, está
dominada en su mayoría por dos filos bacterianos, Bacteroidetes y Firmicutes. La colonización
intestinal por parte de estas bacterias es un proceso multifactorial en los que están implicados,
entre otros, la motilidad y la adherencia a componentes del intestino. La motilidad mediada
por flagelos cumple un rol fundamental en la colonización y virulencia de múltiples bacterias
patógenas, sin embargo, la importancia de la motilidad flagelar en la colonización de bacterias
comensales de la MI, en particular del filo Firmicutes, ha sido pobremente estudiada. El flagelo
permite el movimiento de manera concertada en búsqueda de condiciones favorables en
medios líquidos y semi sólidos, pero además actúa como adhesina a componentes del intestino
como la mucina y células epiteliales. Roseburia inulinivorans (R. inulinivorans) es una bacteria
de la microbiota intestinal de diferentes organismos, flagelada y que pertenece a un género
importante de bacterias en la microbiota intestinal. Los determinantes genéticos que
participan en la colonización de R. inulinivorans no han sido descritos, ya que no existen
herramientas de manipulación genética sitio dirigidas en este género, aunque R. inulinivorans
ha podido ser conjugada con plásmidos derivados de la línea pMTL, convirtiéndola en un buen
candidato para la búsqueda de determinantes genéticos relacionados a la colonización de este
género. Mediante mutagénesis química obtuvimos mutantes sin flagelo de R. inulinivorans
(Fla-
), que posterior a la secuenciación del genoma completo de estas pudimos observar que
las mutaciones generadas afectaban a las proteínas FlhB, FliM y FliP, del cuerpo basal del
flagelo. Estas mutantes in vitro poseen una mayor formación de biopelículas y adherencia a
células epiteliales, y por el contrario una menor adherencia a una capa de mucina. Las
revertantes espontaneas generadas para cada mutante Fla-
(rFla+) confirmaron que los
fenotipos observados se produjeron por las mutaciones en las proteínas descritas
previamente, excepto en FlhB. Interesantemente, las cepas revertantes poseen una mayor
motilidad que la cepa silvestre, lo que podría ser explicado por mutaciones en las proteínas
FliF o CheY, que forman en anillo MS del cuerpo basal o participan la regulación de la
quimiotaxis, respectivamente. En la colonización de ratones no observamos diferencias
significativas entre las cepas mutantes y silvestre, aunque desde las heces solo recuperamos
cepas Fla-
. Por lo tanto, los genes fliM y fliP son funcionales y necesarios en el ensamble del
flagelo en R. inulinivorans y, además, la pérdida de este afecta de forma contraria la
adherencia a las células epiteliales y mucina. Además, si bien no obtuvimos diferencias
significativas en la colonización, observamos una tendencia que indicaría que en ausencia del
flagelo R. inulinivorans favorecería la colonización intestinal.
The human gut microbiota (GM), essential for human health, is dominated by two bacterial phyla, Bacteroidetes and Firmicutes. The intestinal colonization of these bacteria is a multifactorial process involving motility and adherence to intestinal components. Flagellarmotility (FM) plays a vital role in the colonization and virulence of multiple pathogenic bacteria. However, FM's in colonizing commensal bacteria from IM, particularly in the Firmicutes phylum, has been poorly studied. Flagellum all the motility in a concerted way in liquid and semi-solid environments for the search of more favorable conditions but also participates as an adhesin to intestinal components, such as epithelial cells and mucin. Roseburia inulinivorans (R. inulinivorans), is a flagellated member of the IM in different organisms that belongs to an important genus of the GM. Genetics determinants that participate in R. inulinivorans have not been determined yet due to the lack of site-directed mutagenesis tools for this genus. However, R. inulinivorans has been conjugated with pMTL-derived plasmids, which gives this bacterium an advantage over the rest of this genus in searching for genetic determinants related to intestinal colonization. Using chemical mutagenesis, we obtained nonflagellated mutant strains of R. inulinivorans (Fla- ), which were analyzed by whole genome sequencing (WGS) determining that present mutations on FlhB, FliM, and FliP proteins. Flamutants in vitro have a higher biofilm formation and adherence to epithelial cells, and lower adherence to mucin when compared to the wild-type (WT) strain. Spontaneous flagellated revertant (rFla+) strains obtained for each mutant confirm that mutations on each Fla- mutants generate the non-flagellated phenotype, except for FlhB. Interestingly, rFla+ strains have higher motility than WT strains which can be explained by mutations found on FliF or CheY, the MS-ring, or the chemotactic regulatory protein, respectively. We have not observed significant differences between Fla- mutants and WT strain in mice colonization, although we only obtain Fla- mutants and no WT strain bacteria from feces. Therefore, fliM and fliP are functional genes necessary for flagellum assembly in R. inulinivorans, and the loss of this structure affects in a converse way the adherence to epithelial and mucin. Also, even when we did not obtain significant differences in mice colonization, we observed a tendency that could indicate that flagellum absence favors intestinal colonization in R. inulinivorans.
The human gut microbiota (GM), essential for human health, is dominated by two bacterial phyla, Bacteroidetes and Firmicutes. The intestinal colonization of these bacteria is a multifactorial process involving motility and adherence to intestinal components. Flagellarmotility (FM) plays a vital role in the colonization and virulence of multiple pathogenic bacteria. However, FM's in colonizing commensal bacteria from IM, particularly in the Firmicutes phylum, has been poorly studied. Flagellum all the motility in a concerted way in liquid and semi-solid environments for the search of more favorable conditions but also participates as an adhesin to intestinal components, such as epithelial cells and mucin. Roseburia inulinivorans (R. inulinivorans), is a flagellated member of the IM in different organisms that belongs to an important genus of the GM. Genetics determinants that participate in R. inulinivorans have not been determined yet due to the lack of site-directed mutagenesis tools for this genus. However, R. inulinivorans has been conjugated with pMTL-derived plasmids, which gives this bacterium an advantage over the rest of this genus in searching for genetic determinants related to intestinal colonization. Using chemical mutagenesis, we obtained nonflagellated mutant strains of R. inulinivorans (Fla- ), which were analyzed by whole genome sequencing (WGS) determining that present mutations on FlhB, FliM, and FliP proteins. Flamutants in vitro have a higher biofilm formation and adherence to epithelial cells, and lower adherence to mucin when compared to the wild-type (WT) strain. Spontaneous flagellated revertant (rFla+) strains obtained for each mutant confirm that mutations on each Fla- mutants generate the non-flagellated phenotype, except for FlhB. Interestingly, rFla+ strains have higher motility than WT strains which can be explained by mutations found on FliF or CheY, the MS-ring, or the chemotactic regulatory protein, respectively. We have not observed significant differences between Fla- mutants and WT strain in mice colonization, although we only obtain Fla- mutants and no WT strain bacteria from feces. Therefore, fliM and fliP are functional genes necessary for flagellum assembly in R. inulinivorans, and the loss of this structure affects in a converse way the adherence to epithelial and mucin. Also, even when we did not obtain significant differences in mice colonization, we observed a tendency that could indicate that flagellum absence favors intestinal colonization in R. inulinivorans.
Notas
Tesis (Doctor en Filosofía en Biotecnología)
Palabras clave
Microbiota Intestinal, Análisis, Bacterias, Genética, Mutaciones