A Structural Biology approach to fight Antimicrobial Resistance: an X-ray Crystallographic investigation of the antibiotic resistance protein AlbA in complex with pyrrolo[2,1-c][1,4]benzodiazepine compounds

Resistance to antibiotics, primarily caused by their misuse, is a significant and growing threat to global human health. If this situation persists, it is possible for common infections to become increasingly difficult to treat and potentially fatal. In addition to the tragic loss of human lives, this will also result in higher costs for healthcare providers. Furthermore, the rise in antimicrobial resistance jeopardizes many advances in modern medicine, including surgery, chemotherapy, and organ transplantation. We could thus witness a resurgence of bacterial epidemics and pandemics reminiscent of the pre-antibiotic era. To address this problem, it is crucial to gain a scientific understanding of unexplored molecular mechanisms for the development of new antibiotics against critical pathogens. In the context of hospital-acquired infections, the primary targets are a group of multidrug-resistant pathogens collectively known as the ESKAPE pathogens. These include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. Among them, four are Gram-negative species. Xanthomonas albilineans is a xylem-invading bacterial pathogen that causes leaf scald disease in sugarcane. This pathogen produces a mixture of antibacterial compounds, among which the most potent—albicidin—has a broad-spectrum activity at low concentration (as low as 1 ng/mL). The chemical structure of albicidin remained unknown until a decade ago, and the mechanism by which this compound interacts with its main target—the DNA gyrase—has only been elucidated very recently by cryo-electron microscopy (cryo-EM). The Gram-negative Klebsiella oxytoca, often responsible for infections in nursing homes and intensive care units, can, however, evade albicidin's antibacterial action thanks to a protein called AlbA that binds to it with nanomolar affinity. AlbA consists of a ligand binding domain (AlbAS) and a DNA-binding domain. Albicidin binding to AlbAS not only sequesters this compound but also promotes AlbA's activity as a transcriptional activator upregulating itself and likely other antibiotic resistance genes. Sequence alignment and protein structure predictions suggest that AlbA homologues are present in ESKAPE pathogens. In this work, we have investigated the binding of pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) to AlbAS. These are natural compounds produced by Streptomyces species, known for their significant antitumor activity and recently explored also as antibacterial agents. In vitro studies have linked the emergence of resistance to PBDs with the same resistance mechanisms observed for albicidin. In this study, we demonstrate that AlbA is capable of binding to PBDs. We assessed the affinity of the ligands to AlbA using tryptophan fluorescence quenching and found it to be in the low nanomolar range, similar to albicidin. However, differently from albicidin, our biophysical data suggest that AlbA displays two distinct binding sites for the PBDs tested. Using X-ray crystallography, we obtained the structure of AlbAS bound to a PBD ligand at the resolution of approximately 2.5 Å, confirming the existence of two distinct binding sites. These structural findings will pave the way for subsequent structure-based drug design efforts aimed at interfering with the AlbA antibiotic resistance mechanism.