La proteína Omp W de salmonella enterica serovar typhimurium es requerida en el mecanismo de resistencia y expulsión de tóxicos desde la bacteria
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2008
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es
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Universidad Andrés Bello
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Resumen
El transporte a través de la membrana externa (ME) de bacterias Gram negativo
ocurre vía proteínas denominadas genéricamente porinas. Estas se organizan como
homotrímeros formando canales acuosos que permiten el paso de solutos hidrofilicos y de
bajo peso molecular (< 600 Da), siendo el periplasma isoosmótico con el medio
extracelular. Las porinas clásicas presentan entre 12 y 22 hebras tipo B que forman el barril,
a diferencia de las porinas denominadas pequeñas, que presentan entre 8 y 10 hebras tipo B
En 1988 Morimyo aisló y caracterizó mutantes de Escherichia coli sensibles a metil
viológeno. La región del genoma de E. coli ( 4 kpb) que contenía estas mutaciones, ubicada
entre el operón triptofano y el gen tonB, fue utilizada en un estudio comparativo de
genomas y se encontró que presenta pocas variaciones en las diferentes cepas analizadas.
Río abajo del gen tonB se localiza el gen ompW, que codifica para una proteína de
membrana externa con características de porina pequeña.
Resultados preliminares de nuestro laboratorio indican que al igual que ompD, el
gen omp W de Salmonella enterica serovar Typhimurium estaría involucrado en la
resistencia de esta bacteria al tóxico metil viológeno (MV) y permiten especular que
OmpW podría funcionar en conjunto con SmvA en la expulsión de MV y/o de algún otro
producto de desecho derivado del estrés oxidativo.
Evidencias recientes muestran que OmpW de Vibrio cholerae no se comportaría
como una porina clásica al no permitir el ingreso de azúcares en experimentos de hinchado
de liposomas. Este resultado apoyaría la idea de la participación de OmpW en la expulsión
de tóxicos, y no necesariamente en la entrada de sustratos.
Con la finalidad de comparar la susceptibilidad de la cepa silvestre de S.
Typhimurium y sus derivadas mutantes ompW, ompD y ompW ompD, se determinó
su susceptibilidad a MV en placas de medio mínimo suplementado con glucosa. Al igual
que el mutante AsmvA la cepa smvA ompW mostró una sensibilidad a MV 12 veces
mayor que la cepa silvestre. La complementación del mutante doble con el plasmidio
pTOPO-ompW mostró el mismo fenotipo que la mutante simple 8.smvA. La
complementación de células AsmvA !:iomp W con el plasrnidio pGEMT-smvAR mostró una
sensibilidad a MV 1,8 veces mayor en relación a la cepa silvestre. Estos resultados sugieren
que la bomba SmvA utilizaría una vía dependiente de OmpW en la expulsión del tóxico...
The transport across OM happens to proteins named generically porins. The porins organize like homotrimers forming aquous channels that allow the pass of hidrophylic and low molecular weight salutes (<600 Da), being the periplasm isoosmotic with the extracelular environment. The classic porins present between 12 and 22 fibers type B that form the barrel, unlike the minar porins who present between 8 and 1 O fibers type B. In the year 1988, Morimyo isolated and characterized mutants of E. coli sensitive to methyl viologen. This region of the genome of E. coli ( 4 kpb ), located between the tryptophan operan and the tonB gene, was used for a comparative study of genomes and one thought that it presents few variations in the different strains analyzed. Downstream of the tonB gene locates the omp W gene, that it codifies for a protein of outer membrane with characteristics of a minor porin. Preliminary results obtained in our laboratory indicates that, as ompD, the ompW gene of S. Typhimurium also would be involved in the resistance of this bacterium to methyl viologen and we speculate that it might work with SmvA in the efl:lux of this toxic or in the effiux of sorne product of waste derived from the oxidative stress. Recent evidence showed that the Omp W protein from S. Typhimurium would not behave like classic porin, because it <loes not allow the influx of sugars in experiments of liposome swelling. This would support the idea of the participation of this protein in the effiux of toxics, and not necessarily in the entry of substrates. With the purpose of comparing the susceptibility between wild type strain of S. Typhimurium and the mutants: liompW, liompD and the double mutant liompW liompD. The double mutant mvA f:i.ompW was analyzed and a sensibility observed of 12 times major that the wild type strain, this double mutant was possessing the same phenotype of sensibility that the single mutant -1.smvA. When the double mutant was complemented with the pTOPO-ompW plasmid showed the same phenotype that the single mutant mvA and it doubles mvA f:i.omp W. On having fulfilled the complementation of the double mutant mvA f:i.ompW with the pGEMT-smvAR plasmid we obtained sensibility was 1.8 times major with regard to the wild type strain. With these results it is possible to suggest that the pump SmvA would be acting for a route dependent OmpW's in the efllux ofthe toxic...
The transport across OM happens to proteins named generically porins. The porins organize like homotrimers forming aquous channels that allow the pass of hidrophylic and low molecular weight salutes (<600 Da), being the periplasm isoosmotic with the extracelular environment. The classic porins present between 12 and 22 fibers type B that form the barrel, unlike the minar porins who present between 8 and 1 O fibers type B. In the year 1988, Morimyo isolated and characterized mutants of E. coli sensitive to methyl viologen. This region of the genome of E. coli ( 4 kpb ), located between the tryptophan operan and the tonB gene, was used for a comparative study of genomes and one thought that it presents few variations in the different strains analyzed. Downstream of the tonB gene locates the omp W gene, that it codifies for a protein of outer membrane with characteristics of a minor porin. Preliminary results obtained in our laboratory indicates that, as ompD, the ompW gene of S. Typhimurium also would be involved in the resistance of this bacterium to methyl viologen and we speculate that it might work with SmvA in the efl:lux of this toxic or in the effiux of sorne product of waste derived from the oxidative stress. Recent evidence showed that the Omp W protein from S. Typhimurium would not behave like classic porin, because it <loes not allow the influx of sugars in experiments of liposome swelling. This would support the idea of the participation of this protein in the effiux of toxics, and not necessarily in the entry of substrates. With the purpose of comparing the susceptibility between wild type strain of S. Typhimurium and the mutants: liompW, liompD and the double mutant liompW liompD. The double mutant mvA f:i.ompW was analyzed and a sensibility observed of 12 times major that the wild type strain, this double mutant was possessing the same phenotype of sensibility that the single mutant -1.smvA. When the double mutant was complemented with the pTOPO-ompW plasmid showed the same phenotype that the single mutant mvA and it doubles mvA f:i.omp W. On having fulfilled the complementation of the double mutant mvA f:i.ompW with the pGEMT-smvAR plasmid we obtained sensibility was 1.8 times major with regard to the wild type strain. With these results it is possible to suggest that the pump SmvA would be acting for a route dependent OmpW's in the efllux ofthe toxic...
Notas
Tesis (Doctor en Biociencias Moleculares)
Palabras clave
Salmonella Entérica, Expulsión de tóxicos, Transporte de bacterias vía proteínas, Chile