Structure and function studies of lipoprotein signal peptidase II (LspA) from Pseudomonas aeruginosa PAO1 for antibiotic development

Abstract

The standard and most successful treatment for bacterial infections is antibiotic therapy, with drugs such as penicillin. Antibiotics were discovered in 1928 and were successfully implemented clinically to treat bacterial infections by the 1940s. This discovery has saved millions of lives worldwide. Bacteria naturally develop resistance mechanisms to antibiotics, therefore the use of these drugs requires strict control and monitoring to minimize the risk of resistance. However, global overuse and misuse of antibiotics in clinical and agricultural settings has resulted in the emergence of multi-drug resistant (MDR) bacteria. MDR bacteria are resistant to most clinically available antibiotics. Today, antimicrobial resistance is responsible for 700,000 deaths annually world-wide. A 2014 report by the World Health Organization (WHO) predicted that if current trends continue antimicrobial resistance will be responsible for 10 million deaths per year by 2050. This would make it the biggest cause of premature death in the world. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have been identified as being at highest risk of developing MDR resistance. The Gram-negative opportunistic Pseudomonas aeruginosa (P. aeruginosa) is of particular significance in nosocomial acquired infections and in patients with compromised immune systems such as cystic fibrosis (CF). Up to 80 to 95 % of CF patients are killed by respiratory failure caused by chronic bacterial infections. There has been a systemic failure in recent decades in developing novel antibiotics. A new class of drugs for treating Gram-negative bacterial infections, such as P. aeruginosa, has not been developed in over 50 years. New drug targets need to be identified, characterized and understood to facilitate the development of novel antibiotic drugs. The bacterial lipoprotein processing pathway, a three enzyme pathway located at the inner membrane of Gram-negative bacteria, has been identified as a promising target for developing novel antibiotic development. The enzymes of this pathway are essential for cell growth and viability. The focus of this study was the second enzyme of the pathway, lipoprotein signal peptidase II (LspA). Numerous studies have demonstrated that LspA is an essential enzyme in Gram-negative bacteria. Furthermore, two naturally occurring antibiotics, globomycin and myxovirescin, are described in the literature as targeting and inhibiting LspA activity. Unfortunately, these inhibitors are not appropriate for use in the clinic owing to low in vitro potency and poor bioavailability. Chapter 2 of this study describes the recombinant expression, purification, characterisation, in meso crystallization and structural solution of LspA bound to the inhibitor globomycin. The crystal structure was solved using Se-SAD phasing to 2.8 Å resolution. The structure provided insights into the mechanism of globomycin inhibition, substrate binding and catalysis. Chapter 3 describes the cloning, recombinant expression, and purification of the first enzyme in the pathway, diacylglycerol transferase (Lgt). Lgt was used in a coupled assay with LspA which allowed LspA activity to be monitored and to validate conclusions from the crystal structure. Chapter 4 describes the development of two novel SDS-PAGE based assays for monitoring the activity of Lgt and LspA. The LspA activity assay was used in mutagenesis studies to help understand the functional mechanism of the enzyme and to perform a library screen of 28 small compounds that had been predicted to bind LspA by in silico modelling. A single hit compound came out of the screen that inhibited LspA. Chapter 5 describes the development of a high-throughput (HTP) continuous FRET based assay for LspA. The assay was validated for HTP screening and used in a structure activity drug development project. In summary this study has improved the understanding of the function and inhibition of an important drug target, LspA. This study should facilitate structure based drug design and HTP library screening to develop a new generation of antibiotics to help counter the resistance crisis.