Caracterització individual de les sintetases de ppGpp RelA i SpoT) en soques adherents invasives d’Escherichia coli (AIEC )

Justicia Kádár, Àlex
Crohn’s disease is a chronic inflammatory bowel pathology whose ethology is unknown. However, it has been observed the presence of adherent-invasive strain of Escherichia coli (AIEC) in many patients. This pathotype of E. coli can adhere and invade the intestinal epithelial cells besides is capable to survive and replicate within macrophages, triggering the common chronic inflammatory response of Crohn’s disease. The interaction between AIEC and intestinal epithelial cells is mediated by a wide variety of virulence factors regulated by two nucleotides, tetra- and pentaphosphate guanosine ((p)ppGpp). These nucleotides regulate the transcription of wide range of genes involved in the stringent cell response, which occurs in the presence of cellular stress and nutrient starvation, as well as in the infective processes of pathogenic strains. The levels of (p)ppGpp are regulated by RelA and SpoT enzymes in Escherichia coli. The structure of this enzymes is based in a synthetase domain and hydrolase domain, except for RelA, which only has the functional synthetase domain. This project aims to characterize the RelA/SpoT synthetases of (p)ppGpp in AIEC strain and compare them with those of commensal strain MG1655, which is part of the intestinal microbiota. In this study, wild-type strains of MG1655 and LF82 (AIEC) were uses, along with their respective mutant, on deficient in the relA gene (ΔrelA) and another deficient in both relA and spoT (ppGpp0 ). Characterization was carried out through an initial evaluation of the mutation’s effect on bacterial physiology using a nutrient competition assay, growth curves, motility and biofilm formation capability. It was also evaluated the role of ppGpp in the adherent-invasive capacity of AIEC and finally the ppGpp levels were indirectly estimated by qPCR detection of the iraP gene. The results suggest that mutations in relA and spoT do not affect nutrient fitness or specific growth rate but do impact motility and biofilm formation capacity between MG1655 and LF82, suggesting that the role of ppGpp could be determined whether the strain is pathogenic or not. Lastly, the qPCR results indicate a difference in ppGpp levels, and therefore its regulation, between MG1655 and LF82 ​
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