Selecting antibacterial aptamers against an essential outer membrane protein in Pseudomonas aeruginosa via aptamer repurposing approach

Antimicrobial resistance is one of the major threats to global health, resulting in an increasing number of people suffering from severe illness or dying due to infections that were once easily curable with antibiotics. Pseudomonas aeruginosa represents one of the most concerning pathogens involved in antibiotic resistance where the World Health Organisation has classified this gram-negative bacterium as an ESKAPE organism, and it is also listed in the Priority 1: Critical list. Hence, in this project, we opted for a novel intervention by using aptamers to inhibit the growth of Pseudomonas aeruginosa, which can be cost-effective and less laborious. Besides, antimicrobial agents targeting the outer membrane protein (OMP) in Gram-negative bacteria can also be effective at killing or inhibiting bacterial growth as these proteins play an important role in bacteria survival as well as being exposed to the surface, which facilitates direct binding. Aptamer repurposing also reduces the cost and length of new drug development. Unmodified functional DNA aptamers from the Aptagen database were docked with the essential OMP using various docking tools such as HADDOCK 2.4 and HDOCK web server to determine the binding site and binding score of the various aptamers. Aptamers that bind to the active site with a good binding score were synthesised and folded into their 3D structures. P. aeruginosa cells were incubated with the aptamers overnight and the inhibitory effects on the bacterial growth curve were investigated. Apt31 had the best HADDOCK score, and was found to bind near the active site of the OMP. Interestingly, this aptamer also exhibited significant antibacterial activity from the early stationary growth phase onwards. The addition of the aptamer after the late log phase also exhibited a similar effect. Besides, the antibacterial activity of apt31 was also dose-dependent. Hence, we can deduce that apt31 binds to the active site region and blocks the OMP activity. The major role of this OMP is to fold and insert β-barrel proteins into the outer membrane layer. As apt31 hinders the OMP activity, we postulate that the bacteria cells eventually die due to a lack of OMPs, which are essential for survival and virulence. We show that the apt31, an aptamer that binds to an antitumor, is capable of binding to OMP in P.aeruginosa. The aptamer-OMP complex exhibits a significant antibacterial effect in a dose-dependent manner. Future experiments will include the expression of the recombinant OMP and determination of the dissociation constant (Kd) of the aptamer-OMP complex.