Computational and experimental targeting of TraH-TraU interaction in  Acinetobacter baumannii inhibits type IV secretion system (T4SS) mediated transfer of the tetracycline resistance gene

Antimicrobial resistance genes are transferred through conjugation-based horizontal gene transfer, which relies on the type IV secretion system (T4SS) for DNA transfer. Thus, conjugation inhibitors hold promise for controlling the spread of resistance genes among Gram-negative bacteria. Conjugation involves more than twenty proteins encoded by the tra operon. This study focuses on identifying inhibitors that can target critical protein-protein interactions within the conjugative machinery of A. baumannii. Protein-protein interaction analysis, binding energy calculations and dissociation constant (Kd) estimations suggest that the TraH-TraU interaction is stronger than other interactions among the selected Tra proteins. TraH-TraU plays a vital role in DNA transfer and mating pair stabilisation during conjugation and was therefore selected for further investigation. Fifty-eight potential inhibitors were shortlisted based on literature, and their interactions with the TraH-TraU complex were evaluated through molecular docking, Gibbs free energy and Kd calculations, and L42, L50, L53, L55, L56, L57 and L58 were selected. Molecular dynamics simulations further confirmed the stable binding of these compounds with the TraH-TraU complex, which was followed by ADMET analysis and cytotoxicity prediction. Based on that, myristic acid and picolinic acid were selected for further studies, including longer MD simulations. Principal component analysis revealed that myristic acid induces stronger restriction of the essential dynamics of the TraH-TraU complex than picolinic acid, suggesting better inhibition of the conformational flexibility required for conjugative function. Subsequent conjugation assays demonstrated that myristic acid effectively inhibited conjugation at lower concentrations compared to picolinic acid. Scanning electron microscopy (SEM) analysis confirmed that both compounds disrupted the conjugation pilus. Hence, these findings suggest that myristic acid inhibits conjugation-based transfer of resistance genes by targeting the TraH-TraU interaction in Gram-negative bacteria and can be used further to inhibit horizontal gene transfer among bacteria.