Nuevos aspectos de la regulación de la virulencia en cepas enteroagregativas de Escherichia coli

Abstract

En el momento del inicio de esta tesis doctoral, nuestro grupo de investigación, analizando el genoma de la cepa enteroagregativa E. coli 042 pudo comprobar que, además de los genes hha e ydgT (presentes en la mayoría de las enterobacterias), el cromosoma de dicha cepa contenía dos genes muy similares al gen hha, a los que se les denominó hha2 y hha3. También pudo comprobarse que, además del gen hns y su parálogo stpA, la cepa E. coli 042 también codificaba un nuevo parálogo adicional de hns, que se denominó hns2. Una parte de los objetivos de esta tesis doctoral se han centrado en tratar de caracterizar el producto génico de estos nuevos parálogos con el fin de entender qué papel biológico presentan estas copias extra de los genes hha y hns dentro del cromosoma y las vías reguladoras de la bacteria. También en el momento del inicio de este trabajo, el grupo de investigación estaba estudiando el papel de la alarmona (p)ppGpp en la expresión del gen aggR del plásmido pAA2 de la cepa E. coli 042. Tal y como se ha comentado en la Introducción de esta memoria, el producto del gen aggR, la proteína AggR, es un importante regulador de la virulencia bacteriana. Al realizar fusiones transcripcionales del gen aggR con el gen reportero lacZ, situando dicho gen marcador en el extremo 3’ del gen aggR, pudimos observar cómo los clones obtenidos presentaban un fenotipo muy diferente al de la cepa salvaje. Dedicamos una parte de nuestro trabajo a estudiar este fenómeno y los cambios fenotípicos derivados del mismo, lo que la llevado a describir nuevos aspectos de la regulación de la expresión del gen aggR por secuencias localizadas aguas abajo de su pauta abierta de lectura.

The genomes of Gram-negative bacteria encode paralogues and/or orthologues of global modulators. The nucleoid-associated proteins H-NS and Hha are an example: several enterobacteria such as Escherichia coli or Salmonella harbor H-NS, Hha and their corresponding paralogues, StpA and YdgT proteins, respectively. Remarkably, the genome of the pathogenic enteroaggregative E. coli strain 042 encodes, in addition to the hha and ydgT genes, two additional hha paralogues, hha2 and hha3. We show in this work that there exists a strong correlation between the presence of these paralogues and the virulence phenotype of several E. coli strains. hha2 and hha3 predominate in some groups of intestinal pathogenic E. coli strains (enteroaggregative and Shiga toxin- producing isolates), as well as in the widely distributed extraintestinal ST131 isolates. Because of the relationship between the presence of hha2/hha3 and some virulence factors, we have been able to provide evidence for Hha2/Hha3 modulating the expression of the antigen 43 pathogenic determinant. We show that tracking global modulators or their paralogues/orthologues can be a new strategy to identify bacterial pathogenic clones and propose PCR amplification of hha2 and hha3 genes as a virulence indicator in environmental and clinical E. coli isolates. The enteroaggregative E. coli strain 042 also carries, in addition to the hns and stpA genes, a third gene encoding an hns paralogue (termed hns2). This novel H-NS paralogue is widespread within pathogenic strains of the Enterobacteriaceae bacterial family. We present information about the biological function of this novel H-NS paralogue. Compared with H-NS, H-NS2 expression levels are lower and, in addition, its expression exhibits specific features: it is sensitive to the growth temperature and to the nature of the culture medium. Transcriptomic analysis has shown that the H-NS2 protein targets a subset of the genes targeted by H-NS. Genes targeted by H-NS2 correspond mainly with horizontally transferred (HGT) genes and are also targeted by the Hha protein, a fine- tuner of H-NS activity. Growth temperature significantly influences the effect of the H-NS2 protein on the expression of E. coli 042 genes. In addition, we also show that H-NS2 expression can be modified by a reversible C-T transition in the promoter region of the hns2 gene, thus facilitating that increased H-NS2 levels can compensate for H-NS loss.

We also assessed the role of virulence plasmid pAA2 in the pathogenesis of E. coli 042, with special emphasis on the role of the AggR transcriptional regulator. To date, the existing information on the role of this regulator on the virulence of the enteroaggregative strains of E. coli had been obtained mainly by analyzing the effect of mutations in the aggR gene on the overall expression pattern of the 042 strain. Our interest in analyzing the interactions of the pAA2 plasmid with the regulatory networks of the E. coli 042 strain led us to observe an unexpected phenotype, which has allowed us to better understand the role of the AggR regulator. We found that insertion of a genetic sequence in the 3’UTR region of the aggR gene or in the IS element located downstream it results in a notable increase of AggR expression, generating a phenotype that is different from that of the wild type strain (decreased growth rate, increased cellular autoaggregation, increased bacterial motility, increased frequency of pAA2 plasmid conjugation and higher plasmid instability). We also show that higher amounts of AggR protein are correlated with overexpression of genes involved in several metabolic pathways (arginine degradation and β-oxidation of fatty acids), whose products are involved in immunomodulation processes and bacterial pathogenesis in the intestinal tract. We hypothesize that these factors can be related to the ability of strain 042 to colonize the gut. These results demonstrate that in the 3’UTR region of aggR gene are located important genetic sequences involved in post transcriptional regulation of the aggR gene expression levels.