Expression, purification, and preliminary characterisation of the Antibiotic Resistance Effector AlbA from the ESKAPE-class pathogens Acinetobacter baumannii and Pseudomonas aeruginosa

ESKAPE-class pathogens are a critical global health threat due to their robust Multi Drug Resistance (MDR) mechanisms. These pathogens, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., utilise various strategies to evade antibiotic treatments, making infections caused by these bacteria increasingly difficult to treat. Among the diverse resistance mechanisms identified, the naturally occurring protein first identified in K.oxytoca AlbA (albicidin resistance protein) has earned attention for its role in conferring resistance against rigid poly-aromatic natural antibiotic compounds such as albicidin. As highlighted in various studies, AlbA also contributes to this even in ESKAPE pathogens by enhancing survival under antibiotic pressure. AlbA full-length (AlbA-L), starting at the N-terminus, is characterised by a DNA-binding domain (DBD) followed then by a coiled-coil region. At the C-terminus is present a ligand-binding domain (LBD). The latter is also called AlbA short version (AlbA-S). AlbA-L is capable of forming a dimeric structure and interacting with the promoter sequence of its own gene, acquiring a transcription regulator behaviour. This thesis aims to investigate AlbA from two of the most concerning six ESKAPE pathogens: A. baumannii and P. aeruginosa. The recombinant expression, production and purification of the dimeric AlbA-L from both bacteria genera have been documented. Also, the obtaining of P. aeruginosa AlbA-S by site-directed deletion has been documented, as well as its production and purification. Using spectroscopy techniques and with the support of computational prediction, the study characterises AlbA in the presence of several pyrrolobenzodiazepines (PBDs) that are being developed as new antibiotics. Moreover, using fluorescence polarisation (FP) and electrophoretic mobility shift assay (EMSA) techniques, it has been proposed that the interaction between the only current known transcription promoter sequence (which is from K. oxytoca) and the Acinetobacter baumannii AlbA full-length dimer cannot occur. This evidence suggest a new path in order to try to obtain it with greater certainty. Namely, the search for the exact AlbA promoter sequence in other bacterial genera of the ESKAPE class. Furthermore, an investigation through the electron density refinement of an already published "putative" apo AlbA (pdb.6h95) has been pursued, exposing missing details. Apo and co-crystallisation efforts of the Pseudomonas aeruginosa AlbA ligand-binding domain are also present. By doing so, this study aimed to characterise AlbA from these specific pathogens.