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Different Mutations in mucA gene of Pseudomonas aeruginosa mucoid strains in cystic fibrosis patients and their effect on algU gene
expression
Giovana Pulcrano, Dora vita Iula, Valeria Raia, Fabio Rossano, Maria Rosaria Catania
Verónica Arango Baena Alejandra Baldiris Ardila
Third Semester – Molecular biologyUniversidad Pontificia Bolivariana
2012
IntroductionPseudomonas
Bacterium type bacilli.
Gram negative aerobic and anaerobic.
Bacterium with mobility by flagellum.
Easy cultivate in vitro.
IntroductionAeruginosa
Humans opportunistic pathogen that affect immunocompromised and hospitalized patients.
Gram Negative Aerobic with motility single- pole.
The most important diseases caused by P. aeruginosa are lung infections in patients with predisposing local causes such as mechanical ventilation or cystic fibrosis.
IntroductionCystic fibrosis
It is a inherited disease that caused the accumulation of thick, sticky mucus in the lungs, gastrointestinal tract and other body areas.
It is one type of chronic lung disease most common in children and young adults, and is a potentially fatal disorder.
IntroductionGen
Is an ordered sequence of nucleotides in the DNA contain the information necessary for the synthesis of a proteins and RNAs .
Gene mucA
MucA can also act as a regulator of algU transcription forming a
complex with MucB and altering the signal transduction pathway that maintains the low
transcriptional activity of algU
Gene AlgU
algU gene expression was analyzed to determinate if mutations in mucA.
IntroductionThe P. aeruginosa is a gram-negative bacteria of
the genus of Pseudomonas, which mainly affects immunocompromised patients such as cystic fibrosis, the genome of this bacterium has different mutations in the mucA gene affecting the expression of the algU gene.
GENERAL OBJECTIVE.Analysis of the influence of MucA gene
mutations in mucoid strains of P. aeruginosa on algU expression levels designed to establish if different mutations in the mucA gene resulting in length function as a controller of alginate production and swimming ability.
Métodos y Muestra Población de estudio
Se realizo en 119 cepas de P. aeruginosa, a partir de muestras respiratorias de pacientes con fibrosis quísticas. 145 hombres, 155 mujeres con una media de edad de 16.21 años.
La cepa de referencia que se
utilizo fue P. Aeruginosa
ATCC 27853.
Métodos y Muestra Extracción de DNA
El ADN genómico fue evaluado cuantitativamente por espectrofotometría y electroforesis.
La electroforesis es una técnica para la separación de moléculas en un campo eléctrico.
Métodos y Muestra PCR
Para identificar las mutaciones en el gen mucA se utilizo la PCR para obtener unos productos de amplificación.
PCR en tiempo real o cuantitativa
Se utiliza para amplificar y cuantificar de forma absoluta el producto de la amplificación del ADN
Métodos y Muestra Extracción de ARN
Mediante la utilización de ARNasa se hizo una hidrólisis del ARN con el fin de que este no interviniera en los resultados.
La actividad de la DNasa fue bloqueada por la adicción de calor, para desnaturalizarla y posteriormente analizar el resulta espectrofotométricamente.
Métodos y Muestra Movilidad
Este análisis se utilizo para buscar un indicativo de la presencia y función del flagelo.
Si se desplazaba mas de 6 mm era un indicativo positivo.
Métodos y Muestra PRGE ( electroforesis en gel de campos pulsantes)
se utiliza para examinar DNA cromosómico de organismos eucariotas.
en el estudio se utilizo para comparar las cepas y fueron consideradas diferentes si sus patrones de restricción difieren por 7 o mas bandas.
RESULTADOS
Discussions Authors
Publications Agreement or Disagreement
Wood & Ohman
A key elements of alginate operon regulation is the alternative sigma factor AlgU that interacts with MucA and acts as negative regulation of alginate biosynthesis: in fact, the occurrence of mutations in mucA was found to be the most frequent mechanism of conversion to the mucoid form.
Agreement
Boucher et al.
The results of real- time PCR showed that the expression of AlgU gene varied significantly in the three groups. In group II, algU gene expression level were similar to those of reference strains. It can be assumed that in this group, in which algU expression levels are low but there is also production of alginate, gene regulation occurs at different levels.
Agreement
Authors
Publications Agreement or Disagreement
Tart et al
algU was reported to affect not only the amount of alginate production, but also the expression of genes that control flagella functionality. The fleQ gene, involved in the regulation of the synthesis of the flagellum, was inhibited by the expression of the algU gene, thus in each of the strains analyzed a loss mobility inversely proportional to alginate production was expected.
Agreement
Tart et al
The complete inactivation of the flagellum with consequent loss of motility, however, was only observed in the two strains which had mucA novel mutations. In addition, PA91 non- mucoid strain grouped with group II instead of group I in the motility analysis.
Agreement
Discussions
Conclusions The Pathological action is closely related to the
characteristics of the strain P. aeruginosa: flagella, proteins and mucoid phenotype whether or not mucoid.
Conclusions To analyze the sequence of the mucA gene in all
strains was found that 40% (30) were mutated, which tells us that we find great variability of strains in one patient, although the percentage is not too high.
Conclusions
P.aeruginosa to be primarily responsible for respiratory illnesses, so this study on different strains opens the stakes to new clinical treatments.
Conclusions the study showed that alterations in the mucA
gene can alter the expression of the algU gene and thus affect the characteristics of the P.aeruginosa.
Alejandra Baldiris Ardila
Verónica Arango Baena