Duplicaciones génicas y virulencia en Escherichia coli

Autor/a

Bernabeu Lorenzo, Manuel

Director/a

Juárez Giménez, Antonio

Hüttener Queiroz, Màrio

Tutor/a

Juárez Giménez, Antonio

Fecha de defensa

2021-06-08

Páginas

299 p.



Departamento/Instituto

Universitat de Barcelona. Departament de Genètica, Microbiologia i Estadística

Resumen

The pathogenic strains of Escherichia coli can be divided into two large groups: those that cause intestinal infections and those that cause extra- intestinal infections. The strains that belong to the first group are diarrheic and are grouped into pathotypes depending on the virulence factors they present and the strategy they follow to cause an infection. Among the extraintestinal strains it is possible to differentiate those that cause urinary tract infections (UPEC), those that cause neonatal meningitis (NMEC) or those that cause sepsis (SEPEC). The enteroaggregative strains of Escherichia coli (EAEC) represent one of the groups of diarrheal pathogens. EAECs can be distinguished from enteropathogenic strains (EPECs) by their adherence pattern to HEp-2 cells, whereas EPECs present an adherence pattern in the form of microcolonies, EAECs follow a "brick stacking" pattern. This is because enteroaggregative strains express a specific type of adhesive fimbriae that allow adhesion to intestinal cells. Epidemiological studies show that the EAEC strains are genetically heterogeneous, having identified numerous virulence factors in strains of this pathotype. An example of this heterogeneity is the strain O104:H4, which caused an outbreak of bloody diarrhea in Germany in 2011. In addition to the typical characteristics of enteroaggregative strains, the strain O104:H4 exhibits a prophage encoding the Shiga toxin, a well-studied virulence factor characteristic of enterohemorrhagic strains (EHEC). Among the enteroaggregative strains, strain 042 is used as a model for this pathotype, since its genome was sequenced and its virulence factors well characterized. Analyzing the genome of strain 042 we could see that, unlike other strains of Escherichia coli, whose genomes code for the hha gene and its ydgT paralog, its chromosome encodes four paralogs of the hha gene, which are hha, ydgT, and the new hha2 and hha3 that had not been described until now. We hypothesize that the presence of these new paralogs hha2 and hha3 in strain 042 could be associated with the presence of genes related to virulence. Through genomic analysis it was possible to establish the relationship between the presence of new paralogs of the Hha protein with the yeeR and irmA genes. Furthermore, due to the presence of a higher number of copies of the Hha regulator and other genes such as, for example, flu, we decided to study, using bioinformatic tools, the existence of duplicate genes and their distribution in different pathotypes. In this way, we stablish gene duplication patterns in all pathotypes and possible relationships between them. Owing to the irmA gene is duplicated in strain 042 and associated with the presence of the hha2/hha3 genes, the expression of both copies (irmA2244 and irmA4509) was characterized under different conditions. As a result, the expression of the IrmA4509 copy is higher in minimal medium (M9) at 37 ºC. Furthermore, the expression of this copy increases under conditions of limitation of the carbon source (glucose) but is reduced in the absence of oxygen. On the other hand, the expression of the IrmA2244 copy is very low or null in all the conditions studied. Next, the regulation of both copies was studied, determining the role of the H-NS/Hha system and the OxyR/Dam system. In this case, the IrmA4509 copy is overexpressed in the absence of hha and, above all, in the absence of hha and hha2 (hhanull), and in the absence of the OxyR regulator. However, no expression could be detected from the copy Irma2444. Finally, the position of the promoter region of both copies was determined by 5' RACE, observing nucleotide changes between them. A directed mutagenesis assay was carried out, by which the importance of these nucleotide changes in the expression of both copies was determined. On the other hand, the possible role in virulence of a region of duplicated genes widely distributed among all pathotypes was studied. For this, biofilm formation and infectivity tests were performed in the Galleria mellonella model organism, obtaining relevant results.

Palabras clave

Bacteriologia; Bacteriología; Bacteriology; Escheríchia coli; Escherichia coli; Etiologia; Etiología; Etiology; Genètica molecular; Genética molecular; Molecular genetics; Duplicació de l'ADN; Replicación del ADN; DNA replication

Materias

577 - Bioquímica. Biología molecular. Biofísica

Área de conocimiento

Ciències Experimentals i Matemàtiques

Documentos

MBL_TESIS.pdf

8.749Mb

 

Derechos

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