Discovery of Antibiotic Candidates Targeting the LPS Transport System in Pseudomonas aeruginosa

Abstract

Antibiotic resistance is one of the most urgent public health crises worldwide. As bacterial pathogens develop resistance to existing antibiotics, fewer treatment options remain available for curing deadly infections. Pseudomonas aeruginosa is an opportunistic Gram-negative bacterial pathogen that is difficult to treat with antibiotics and is associated with life-threatening infections in cystic fibrosis patients. Patients with chronic P. aeruginosa infections have a doubled mortality rate over patients who don’t, which increases to eightfold when the Pseudomonas strain is classified as multidrug-resistant. P. aeruginosa contains a group of proteins called the Lipopolysaccharide (LPS) Transport System that transports LPS from the inner membrane to the cell’s outer membrane. LPS is essential for protecting bacterial cells from environmental stress, maintaining the integrity and structure of the outer membrane, and is associated with antibiotic resistance. In P. aeruginosa, LptH is a vital component of the LPS Transport assembly. This protein oligomerizes to form a bridge between the inner and outer membranes, facilitating transport of LPS. It is essential for the viability of P. aeruginosa cells and, therefore, a promising drug target. Terminating LPS transport using inhibitors of LptH would make E. coli cells more susceptible to the host’s innate immune system and antibiotics used in clinical settings. This project aims to characterize LptH and identify antibiotic candidates that can terminate LptH-facilitated transport of LPS, making the outer membrane of the cell more susceptible to other antibiotics. To test the impact of LptH expression on the efficacy of existing membrane-targeting drugs, an LptH antibiotic screening strain was developed using a pBAD24 cloning vector, allowing for a tightly controlled expression of this protein. Preliminary antibiotic susceptibility assays using this strain revealed lower susceptibility to membrane-targeting antibiotics when overexpressing LptH. An expression strain was constructed to enable in vitro biochemical studies, and LptH was successfully overexpressed and purified under native conditions. Preliminary binding affinity experiments demonstrated that E. coli LPS extracts bound to LptH with an estimated dissociation constant of 90 mM. Future experiments include the assessment of the binding affinity for a computationally identified library of compounds against purified LptH. Gene complementation experiments will be used to develop an E. coli strain lacking LptA, a homolog of LptH, to more accurately determine the inhibitory effects of these compounds on LptH using cell-based assays.