Untersuchung der Bedeutung des uncharakterisierten Gens ygfB für die β-Laktamresistenz von multi-resistenten Pseudomonas aeruginosa Stämmen
Zusammenfassung der Projektergebnisse
Data prior to this study revealed that the uncharacterized protein YgfB contributes to βlactam resistance in the multi-drug resistant P. aeruginosa strain ID40 by increasing ampC expression via an unknown mechanism. The main goal of the presented study was to identify how YgfB mechanistically contributes to β-lactam resistance in P. aeruginosa. We could demonstrate that YgfB represses expression of the amidase AmpDh3. The amidase AmpDh3 was determined to be located in the cytoplasm and can cleave the peptidoglycan recycling products anhydro-MurNAc-peptides into anhydro-MurNAc and peptide. The YgfB mediated repression of AmpDh3 results in higher amounts of anhydro-MurNAc-peptides. Anhydro-MurNAc-peptides bind to the transcriptional activator AmpR. This leads to derepression of AmpR and expression of the β-lactamase AmpC as well as other genes. High βlactamase activity leads to advanced cleavage of β-lactam antibiotics and consequently increased resistance to β-lactam antibiotics. The ampDh3-TUEID40-01945 operon and the alpBCDE operon are transcriptionally regulated by the anti-terminator AlpA. AlpA binds to the AlpA binding elements (ABE) of these promoters and subsequently to the RNA polymerase (RNAP). These interactions allow the RNAP to pass the terminator signal in the promoter and leads to ampDh3 transcription. We could demonstrate that YgfB directly interacts with AlpA. The binding of YgfB prevents the binding of AlpA to the ABE and thereby represses AlpA mediated ampDh3 expression. Taken together, we could elucidate the complete pathway how YgfB contributes to increased β-lactam resistance. Investigation of the impact of YgfB on ampDh3 expression and β-lactam resistance in several P. aeruginosa strains leads to the assumption that the findings can be generalized for P. aeruginosa. Induction of ampDh3 expression can be achieved by DNA damage induced for instance by ciprofloxacin. Ciprofloxacin leads to higher AlpA production and higher AmpDh3 abundance. Deletion of ygfB further increases ampDh3 expression upon Ciprofloxacin treatment but does not affect AlpA levels. Moreover, deletion of ygfB increases the sensitivity to ciprofloxacin βlactam combination treatment. Interestingly while ygfB orthologs are found in many γ-proteobacteria, AlpA or AmpDh3 orthologs are rarely found in other species beside Pseudomonas. This means that the YgfB repression of ampC expression seems to be rather a unique feature of Pseudomonas. This led to the question what might be general functions of YgfB. To address the function of YgfB in E. coli transcriptome analyses were performed comparing gene expression of an E. coli strain and a ygfB deletion mutant. We could not observe any transcriptional changes upon ygfB deletion, indicating that the YgfB mediated repression of gene expression might be a unique feature of P. aeruginosa. Therefore, we wondered whether beside AlpA there might be other proteins YgfB interacts with. Screening for interaction partners of E. coli and P. aeruginosa YgfB revealed so far that YgfB directly interacts with the small subunit ribosomal protein uS5 (RpsE). How and whether this interaction modulates ribosomal assembly or translation is so far elusive.
Projektbezogene Publikationen (Auswahl)
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YgfB increases β-lactam resistance in Pseudomonas aeruginosa by counteracting AlpA-mediated ampDh3 expression. Communications Biology, 6(1).
Eggers, Ole; Renschler, Fabian A.; Michalek, Lydia Anita; Wackler, Noelle; Walter, Elias; Smollich, Fabian; Klein, Kristina; Sonnabend, Michael S.; Egle, Valentin; Angelov, Angel; Engesser, Christina; Borisova, Marina; Mayer, Christoph; Schütz, Monika & Bohn, Erwin
