Tesis doctoral de Anais Panosa BorrÁ s
Salmonella enterica serovar typhimurium (s. Typhimurium) is a gram negative intracellular human pathogen causing gastroenteritis in humans as well as a systemic infection in mice similar to human typhoid fever (which is caused by s. Typhi). One of the main features of s. Enterica infection is its capacity to actively invade epithelial cells and proliferate inside macrophages. s. Typhimurium presents three classes of ribonucleotide reductases (rnrs) in its genome: class ia, class ib and class iii. Rnrs are essential enzymes because they carry out the de novo synthesis of deoxyribonucleotides (dntps), needed for dna synthesis and repair, by reducing ribonucleotides (ntps). Up to date, three different classes of rnrs have been described, differing in their mechanism of radical generation, their three-dimensional structure, allosteric regulation and their oxygen dependence. Nevertheless, they all have in common the mechanism of reaction and the use of an organic free radical to initiate catalysis. this work has studied the transcriptional regulation of the three rnr classes present in s. Typhimurium, giving importance to two main regulators: nrdr and fur. We have studied the effects of nrdr deletion in each class of rnr gene expression. Our results indicate that nrdr is a repressor of all three classes, but it shows a stronger repression when regulating nrdhief expression. We have also described nrdr recognition sites (nrdr boxes) and we have obtained mutant proteins in some residues that affect nrdr functionality in vivo, possibly due to their participation in datp/atp union. mutation of fur causes an upregulation of nrdhief expression up to five fold compared to the wild type strain. We have detected a putative fur recognition sequence within the nrdhief promoter region. Mutation of this recognition sequence causes an upshift in nrdhief expression similar to the level observed in the fur mutant. Using electrophoretic mobility shift assays (emsa) we have confirmed that fur interacts with the nrdhief promoter region. under normal conditions, s. Typhimurium uses class ia rnr to de novo synthesize dntps in the presence of oxygen. Class ia is essential for normal growth and class ib is not able to complement its mutation unless an extra copy of nrdhief is introduced. The presence of two rnrs with redundant activities in the same organism, the fact that other bacterial species use class ib for dntp synthesis in then presence of oxygen, and that both e. Coli and s. Typhimurium have retained class ib enzymes during evolution, suggest that this class might be expressed under specific growth conditions. we have studied the role of rnrs in the virulence of s. Typhimurium sl1344 by means of different mutants and double mutants in each rnr class as well as mutants in their transcriptional regulators (fur, nrdr). Infection assays performed in macrophage cell lines and epithelial cell lines have allowed to elucidate which rnr is responsible for s. Typhimurium growth during infection. Our results indicate that class ia is the rnr responsible for invasion and proliferation inside macrophages, while class ib and class iii do not seem to be essential. However, class ia mutants overexpressing class ib are capable of surviving the first hours of infection (2-6 h). We think that is in this first stage of the infection process (when the oxidative burst takes place), specific conditions generate and activate nrdhief expression. It is possible that hydrogen peroxide concentrations are responsible for the inhibition of the fur repressor activity. The highest demand of dntps due to dna lesions makes it necessary for an extra dntp supply that will be provided by the nrdef enzyme.
Datos académicos de la tesis doctoral «Ribonucleotidil reductases de salmonella enterica serovar typhimurium: regulació transcripcional i participació en la patogénesi«
- Título de la tesis: Ribonucleotidil reductases de salmonella enterica serovar typhimurium: regulació transcripcional i participació en la patogénesi
- Autor: Anais Panosa BorrÁ s
- Universidad: Autónoma de barcelona
- Fecha de lectura de la tesis: 26/02/2009
Dirección y tribunal
- Director de la tesis
- Ignasi Roca Subir?
- Tribunal
- Presidente del tribunal: Antonio Villaverde corrales
- albert Jordan valles (vocal)
- (vocal)
- (vocal)