Login
Communauté Vinci
Extérieur
Si votre nom d'utilisateur ne se termine pas par @vinci.be ou @student.vinci.be, utilisez le formulaire ci-dessous pour accéder à votre compte de lecteur.
Titre : | Le rôle du polyphosphate dans la résistance aux aminoglycosides et dans la biogenèse du ribosome chez Escherichia coli. |
Auteurs : | Helen Macki, Auteur ; François Beaufay, Promoteur |
Type de document : | Travail de fin d'études |
Editeur : | Woluwe-Saint-Lambert : Haute École Léonard de Vinci, 2022 |
Langues: | Français |
Index. décimale : | TFE - Biologie médicale |
Mots-clés: | herichia coli K-12 ; polyphosphate ; sous-unités du ribosome ; aminoglycosides ; Biogenèse du ribosome |
Résumé : |
Introduction: The use and development of antibiotics in the mid-20th century contributed to the increase in human life expectancy. From the 1980s, antibiotic-resistant strains of bacteria began to spread. These resistances became a public health issue for scientists investing in the exploration of solutions to treat infectious diseases, where the study of polyphosphate (PolyP) metabolism is of interest. PolyP is an inorganic phosphate polymer, present in all species, but it has been mainly studied in prokaryotes where it has been particularly associated with stress resistance mechanisms. The laboratory has previously shown that the bacterial strain Escherichia coli K-12 MG1655 unable to synthesize the PolyP, has an increased sensitivity to aminoglycosides as well as committing errors during translation at the ribosome level by acting specifically on the 16S rRNA of the 30S subunit of the ribosome, also in its fidelity. The objective of this research is to identify the role of polyphosphate in aminoglycoside resistance and ribosome biogenesis in E. coli K-12 MG1655 using two approaches. The first one is to use candidates/genes (Hfq, Lon, DnaK/J and GroEL/ES) involved in ribosome biogenesis that are related to PolyP to observe a genetic interaction between ppk and these candidates. The Hfq factor is involved in the maturation of 16S ribosomal RNA and Lon is responsible for the degradation of ribosomal subunits. The second one is to find sequences capable of complementing defects in the Δppk mutant strain in the face of aminoglycoside by using a pBAD33 overexpression library containing fragments of the E. coli genome.
Methods: In vitro, plasmid extraction was done by mini-prep. Candidate deletion was performed by inserting aminoglycoside resistance cassette using the Lambda Red PCR system. The excision of the resistance cassette is done by electroporation following the transformation of the mutant with the plasmid pCP20. The preparation of electro-competent cells and their transformation into a Δppk mutant strain. The transduction technique was done to obtain a double deletion and to observe a genetic interaction between the PolyP and the candidates. Results: Regarding the first approach, combining the Δhfq and Δlon mutants with the Δppk mutant showed the presence of an additive effect, suggesting that these two genes act in two parallel ways with ppk. Furthermore, overexpression of both chaperones DnaK/J and GroEL/ES in the Δppk mutant showed that there was no compensation for defects in a Δppk mutant via these chaperones in the presence of Streptomycin. The second approach showed us that the Δppk pBAD33 library is ready to be spread on media containing different concentrations of antibiotics such as Gentamycin 0.25-0.3μg/mL and Erythromycin 100-150μg/mL. Conclusion: The additive effect of lon and ppk deletions, suggests that PolyP is not involved in ribosome subunit recycling. Similarly, the additive effect of the hfq and ppk mutations suggests that PolyP regulates ribosome biogenesis independently of Hfq. Thus, the PolyP does not affect ribosome biogenesis via either Hfq and Lon actors. PolyP and the chaperone foldase systems DnaK/J and GroEL/ES dont have redundant functions in ribosome biogenesis. Thus, several strategies are still in progress to find the role of polyphosphate in ribosome biogenesis by testing other genes as well as pursuing the second approach and finding the surviving candidates to extract their plasmid and send it to sequencing, in order to find the sequence that will be able to compensate for the defects in a Δppk mutant strain and provide insights on the role of PolyP. |
Accès : | Identifiez-vous avant d'accéder au document électronique |
Disponible en ligne : | Oui |
Lieu du stage : | Institut de Biologie et de Médecine Moléculaire (IBMM) - ULB Gosselies |
Département : | Biologie médicale |
Documents numériques (1)
Ce document n'est visible qu'après identification
TFE - Biologie médicale Adobe Acrobat PDF |